Richard G Dorrell
email: dorrell@bio.ens.psl.eu

Fuhai Liu

Chris Bowler

Chapter 6: Reconstructing dynamic evolutionary events in diatom nuclear and organelle genomes

Keywords: horizontal gene transfer, endosymbiosis, shopping bag model, cryptomonads, haptophytes, dinoflagellates

The diatoms evolved within the stramenopiles, an ecologically important and diverse assemblage of eukaryotes that includes both photosynthetic macrophytes and microalgae, as well as non-photosynthetic heterotrophs and parasites. The evolutionary history of the stramenopiles, which stretches back to the Palaeozoic, has been marked by the acquisition of chloroplasts in a recent common ancestor of their photosynthetic members, the ochrophytes; and progressive gains of genes in the nuclear genome by horizontal and endosymbiotic gene transfer. Here, we place diatoms in their actual evolutionary context within the stramenopiles; identify gene transfers that have shaped the coding content of the diatom nucleus; and profile sources of differences in chloroplast and mitochondrial genome content between different stramenopiles including diatoms. We underline the importance of considering diatoms as evolutionary mosaics, supported by genes of bacterial, red, green and other eukaryotic algal origin as illustrated by multiple phylogenomic studies realised over the last two decades; and the relatively limited changes to organelle genome content in diatoms compared to other stramenopile lineages. However, we identify a previously undocumented transfer of a novel open reading frame of the chloroplasts of green algae into the ochrophytes, underlining the importance of changes in organelle and nuclear gene content, in defining the current biology of diatoms.

Table of Contents

The evolutionary context of diatoms within the stramenopiles ……………………… 4

Diversity of photosynthetic and non-photosynthetic stramenopile groups …………………... Fig. 1. Evolutionary positions of diatoms within the eukaryotes and the stramenopiles …….. Phylogenetic arrangement and endosymbiotic histories of the stramenopiles .……………… 5 2. Phylogenomic insights into diatom evolution ………………………………………...

The evolutionary context of diatoms within the stramenopiles

Diversity of photosynthetic and non-photosynthetic stramenopile groups

Diatoms belong to the stramenopile phylum, which sits within the "SAR clade" (Stramenopiles, Alveolates and Rhizaria) supergroup of the tree of life (2, 6, 7) (Fig. 1). Besides stramenopiles, the SAR clade contains the alveolates (including dinoflagellates, which are the principal photosynthetic component of corals, and apicomplexans such as the malaria parasite Plasmodium), and rhizarians (including the important fossil markers, Fig . 1. Evolutionary positions of diatoms within the eukaryotes and the stramenopiles. Top : a schematic tree of eukaryotic diversity, adapted from (5) ; bottom, a closeup of stramenopile lineages, adapted from [START_REF] Dorrell | Secondary plastids of stramenopiles[END_REF], showing the global and local evolutionary context of diatoms. Cell images are reprinted from Encyclopedia of Life (www.eol.org), Prof. Connie Lovejoy (Université Laval), and Dr. Zhanru Shao (Institute of Oceanology, Chinese Academy of Sciences), with permissions. are observed in most phyla, and cryptomonads and haptophytes share a derived, bacterial isoform of the chloroplast-targeted protein rpl36. Cryptomonads, picobiliphytes and kathablepharids are believed to be closely related; there are conflicting data regarding the phylogenetic positions of haptophytes, centrohelids and telonemids. No parasitic taxa are known. A genome sequence is available for the haptophyte Emiliania huxleyi; a sequence for the cryptomonad Guillardia theta is in preparation.

Cryptomonads: Mostly autotrophic cells distinguished by characteristic arrays of bipartite flagellar hairs, a geometric cell coat and flattened mitochondrial cristae. The secondary, red algalderived chloroplasts retain a nucleomorph (relict algal nucleus). Cryptomonad-derived plastids have been observed in dinoflagellate species; these may be tertiary endosymbionts, or may be obtained by kleptoplastidy. Genome sequences: Guillardia theta (in progress).

Kathablepharids: Free-living heterotrophic flagellates, with a distinctive, spiralled organic sheath, complex conical feeding apparatus and a peripheral ER. Feeding may occur via engulfment or myzocytotic consumption of prey cytoplasm and individuals may swarm to engulf prey. Five genera are currently known; none have true chloroplasts, although one species, Hatena, exists with a green algal photosymbiont.

Picobiliphytes: Small (6 μm long) planktonic organisms of unknown general appearance, distributed throughout sub-arctic, temperate and tropical waters, described only from environmental studies. Picobiliphytes have an organelle similar in fluorescence profile to the plastids of red algae and cryptomonads, and have been suggested to retain a nucleomorph.

Haptophytes: Free-living mixotrophic or autotrophic flagellates, distinguished by a haptonema (a locomotory, attachment and feeding organelle supported by microtubules), and distinctive lamellae in the red algal-derived chloroplasts; species may be naked or covered in calcareous, organic or in one case siliceous scales. Several species (e.g. Phaeocystis globosa) are agents of fish-killing

Haptophytes Chlorarachniophytes Euglenids Dinoflagellates

synapomorphies have been reported, although similar flagella, sub-lamellar vesicles and ejectisomes are observed in most phyla, and cryptomonads and haptophytes share a derived, bacterial isoform of the chloroplast-targeted protein rpl36. Cryptomonads, picobiliphytes and kathablepharids are believed to be closely related; there are conflicting data regarding the phylogenetic positions of haptophytes, centrohelids and telonemids. No parasitic taxa are known. A genome sequence is available for the haptophyte Emiliania huxleyi; a sequence for the cryptomonad Guillardia theta is in preparation.

Cryptomonads: Mostly autotrophic cells distinguished by characteristic arrays of bipartite flagellar hairs, a geometric cell coat and flattened mitochondrial cristae. The secondary, red algalderived chloroplasts retain a nucleomorph (relict algal nucleus). Cryptomonad-derived plastids have been observed in dinoflagellate species; these may be tertiary endosymbionts, or may be obtained by kleptoplastidy. Genome sequences: Guillardia theta (in progress).

Kathablepharids: Free-living heterotrophic flagellates, with a distinctive, spiralled organic sheath, complex conical feeding apparatus and a peripheral ER. Feeding may occur via engulfment or myzocytotic consumption of prey cytoplasm and individuals may swarm to engulf prey. Five genera are currently known; none have true chloroplasts, although one species, Hatena, exists with a green algal photosymbiont.

Picobiliphytes: Small (6 μm long) planktonic organisms of unknown general appearance, distributed throughout sub-arctic, temperate and tropical waters, described only from environmental studies. Picobiliphytes have an organelle similar in fluorescence profile to the plastids of red algae and cryptomonads, and have been suggested to retain a nucleomorph.

Haptophytes: Free-living mixotrophic or autotrophic flagellates, distinguished by a haptonema (a locomotory, attachment and feeding organelle supported by microtubules), and distinctive lamellae in the red algal-derived chloroplasts; species may be naked or covered in calcareous, organic or in one case siliceous scales. Several species (e.g. Phaeocystis globosa) are agents of fish-killing forams). The group is distantly related both to plants and to animals, sharing a last common ancestor with each no more recently than one billion years ago [START_REF] Parfrey | Estimating the timing of early eukaryotic diversification with multigene molecular clocks[END_REF]. The stramenopiles themselves are evolutionarily ancient, radiating at least six hundred million years before the present [START_REF] Brown | A molecular genetic timescale for the diversification of autotrophic stramenopiles (Ochrophyta): substantive underestimation of putative fossil ages[END_REF], placing them on a common level of antiquity to plants and animals [START_REF] Dorrell | Do red and green make brown?: Perspectives on plastid acquisitions within chromalveolates[END_REF].

The stramenopiles are an ecologically diverse, and environmentally important group of eukaryotes. They are characterised, in particular, by their flagellar organisation, which typically includes one short flagellum, and one long flagellum covered in tubular « hair »-like structures; with the uneven length of their flagella giving rise to an alternate name of « heterokonts » [START_REF] Adl | The revised classification of eukaryotes[END_REF][START_REF] Cavalier-Smith | A revised six-kingdom system of life[END_REF]. Beyond this, stramenopiles contain few conserved features, although their monophyly is well supported by molecular data [START_REF] Burki | Phylogenomics reshuffles the eukaryotic supergroups[END_REF][START_REF] Elias | The RAB family GTPase Rab1A from Plasmodium falciparum defines a unique paralog shared by chromalveolates and Rhizaria[END_REF].

Alongside the diatoms, the stramenopiles contain numerous other photosynthetic taxa, referred to collectively as "ochrophytes" (Fig. 1). These include, but are not limited to: giant macrophytic kelps (found within the phaeophytes), which can grow to nearly one hundred meters in length [START_REF] Fork | Light energy distribution in the brown alga Macrocystis pyrifera (giant kelp)[END_REF], and unicellular and colonial algae (e.g., within the chrysophytes, or "golden algae", dictyochophytes, or "silicoflagellates", and pelagophytes), which can have cell diameters of only a couple of micrometers. Several other stramenopile groups (particularly phaeophytes, dictyochophytes and chrysophytes) are important contributors to marine and freshwater photosynthesis (5) Some of the ochrophyte groups are silicifying, like diatoms (e.g., silicoflagellates within the dictyochophytes; synurophytes within the chrysophytes), while others are not [START_REF] Dorrell | Secondary plastids of stramenopiles[END_REF][START_REF] Ichinomiya | Diversity and oceanic distribution of the Parmales (Bolidophyceae), a picoplanktonic group closely related to diatoms[END_REF][START_REF] Hendry | Competition between silicifiers and non-silicifiers in the past and present ocean and its evolutionary impacts[END_REF]. Some of these photosynthetic groups are also known to engage in mixotrophic strategies, either through the phagocytotic consumption of bacteria (e.g., in chrysophytes and dictyochophytes [START_REF] Graupner | Evolution of heterotrophy in chrysophytes as reflected by comparative transcriptomics[END_REF][START_REF] Walker | Eukaryotic systematics: a user's guide for cell biologists and parasitologists[END_REF]), or through the osmotrophic uptake of extracellular organic nutrients (e.g., in diatoms and pelagophytes) [START_REF] Villanova | Investigating mixotrophic metabolism in the model diatom Phaeodactylum tricornutum[END_REF], while in others (e.g., kelps) neither strategy has been reported (2). Moreover, there is a substantial non-photosynthetic component to stramenopile diversity (Fig. 1). These include some lineages that resolve within otherwise photosynthetic groups, e.g., non-photosynthetic diatoms [START_REF] Kamikawa | Multiple losses of photosynthesis in Nitzschia (Bacillariophyceae)[END_REF][START_REF] Pendergrass | The genome of the nonphotosynthetic diatom, Nitzschia sp.: Insights into the metabolic shift to heterotrophy and the rarity of loss of photosynthesis in diatoms[END_REF], dictyochophytes [START_REF] Sekiguchi | Vestigial chloroplasts in heterotrophic stramenopiles Pteridomonas danica and Ciliophrys infusionum (Dictyochophyceae)[END_REF][START_REF] Kayama | Highly reduced plastid genomes of the non-photosynthetic dictyochophyceans Pteridomonas spp. (Ochrophyta, SAR) are retained for tRNA-Glubased organellar heme biosynthesis[END_REF], and chrysophytes [START_REF] Graupner | Evolution of heterotrophy in chrysophytes as reflected by comparative transcriptomics[END_REF][START_REF] Dorrell | Principles of plastid reductive evolution illuminated by nonphotosynthetic chrysophytes[END_REF], and accordingly retain leucoplasts (non-photosynthetic plastids). Other non-photosynthetic stramenopile groups contain no photosynthetic members and no trace of plastids. These "aplastidic stramenopiles" include important pathogens: e.g., oomycetes, parasites of algae and plants, such as the causative agents of potato blight and sudden oak death [START_REF] Stiller | Are algal genes in nonphotosynthetic protists evidence of historical plastid endosymbioses?[END_REF][START_REF] Levesque | Genome sequence of the necrotrophic plant pathogen Pythium ultimum reveals original pathogenicity mechanisms and effector repertoire[END_REF]; labyrinthulomycetes, which are important pathogens of marine algae and invertebrates [START_REF] Tsui | Labyrinthulomycetes phylogeny and its implications for the evolutionary loss of chloroplasts and gain of ectoplasmic gliding[END_REF]; and Blastocystis, a human gut commensal which may have harmful effects in immunodeficient hosts [START_REF] Eme | Lateral gene transfer in the adaptation of the anaerobic parasite Blastocystis to the gut[END_REF]. In addition, the aplastidic stramenopiles include important marine saproptrophs (e.g., hypochytriomycetes, related to oomycetes [START_REF] Leonard | Comparative genomic analysis of the 'pseudofungus' Hyphochytrium catenoides[END_REF]) and predator groups (e.g., bicosoecids, related to Blastocystis (36)).

Phylogenetic arrangement and endosymbiotic histories of the stramenopiles

The phylogenetic relationships within the stramenopiles have been revealed progressively by morphology, electron microscopy and biochemistry [START_REF] Adl | The revised classification of eukaryotes[END_REF][START_REF] Cavalier-Smith | A revised six-kingdom system of life[END_REF], alongside single-gene, and subsequently multigene phylogenies (4, 6, 37) (Fig. 1). The exclusively non-photosynthetic groups form the most basally divergent stramenopile nodes, with oomycetes forming the closest major sister-group to the photosynthetic ochrophytes (6, 37) (Fig. 1). Within the ochrophytes, three major groups are apparent (Fig. 1) : the "chrysista", which include phaeophytes; chrysophytes, and the oil-producing alga Nannochloropsis [START_REF] Ichinomiya | Diversity and oceanic distribution of the Parmales (Bolidophyceae), a picoplanktonic group closely related to diatoms[END_REF][START_REF] Rodolfi | Microalgae for oil: strain selection, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor[END_REF]; the « hypogyristea», containing pelagophytes and dictyochophytes [START_REF] Ichinomiya | Diversity and oceanic distribution of the Parmales (Bolidophyceae), a picoplanktonic group closely related to diatoms[END_REF][START_REF] Dorrell | Chimeric origins of ochrophytes and haptophytes revealed through an ancient plastid proteome[END_REF]; and the "khakista" or diatoms, and their immediate sister group, a single-genus group alternatively labelled Bolidomonas or Triparma [START_REF] Ichinomiya | Diversity and oceanic distribution of the Parmales (Bolidophyceae), a picoplanktonic group closely related to diatoms[END_REF][START_REF] Tajima | Sequencing and analysis of the complete organellar genomes of Parmales, a closely related group to Bacillariophyta (diatoms)[END_REF]. The hypogyristea and khakista are clearly sister-groups, whereas the monophyly of chrysista is uncertain, with some phylogenomic studies resolving the constituent lineages as a single group [START_REF] Derelle | A phylogenomic framework to study the diversity and evolution of stramenopiles (heterokonts)[END_REF]; and others indicating a basally divergent group of pinguiophytes, synchromophytes, synurophytes and chrysophytes, which precede the divergence of phaeophytes, raphidophytes and eustigmatophytes from the remaining ochrophytes [START_REF] Dorrell | Phylogenomic fingerprinting of tempo and functions of horizontal gene transfer within ochrophytes[END_REF][START_REF] Burki | Untangling the early diversification of eukaryotes: a phylogenomic study of the evolutionary origins of Centrohelida, Haptophyta and Cryptista[END_REF].

Chloroplasts originate via the endosymbiotic uptake of free-living photosynthetic bacteria (in the case of primary endosymbiosis), or of single-celled eukaryotic algae containing chloroplasts of red or green origin (in the case of secondary endosymbiosis; Fig. 1). The engulfed cells are converted into stable, intracellular organelles; and this process has been observed to occur in multiple algal groups across the tree of life (Fig. 1) [START_REF] Walker | Eukaryotic systematics: a user's guide for cell biologists and parasitologists[END_REF]. The chloroplasts found in ochrophytes are typically characterised by the presence of four surrounding membranes, the outermost of which is contiguous to the endoplasmic reticulum [START_REF] Ishida | Endomembrane structure and the chloroplast protein targeting pathway in Heterosigma akashiwo (Raphidophyceae, Chromista)[END_REF], and possess chlorophyll c as a light harvesting pigment [START_REF] Kowallik | The chloroplast genome of a chlorophyll a+c-containing alga, Odontella sinensis[END_REF], although exceptions to these paradigms are known (2, 44) (Fig. 2). Both of these features are characteristic of chloroplasts acquired through the secondary endosymbioses of red algae (i.e., those found in cryptomonads, haptophytes, dinoflagellates, and photosynthetic apicomplexans; Fig. 1), although are not known in red algae themselves [START_REF] Dorrell | Secondary plastids of stramenopiles[END_REF]. In contrast, ochrophytes do not retain phycobiliprotein subunits, which are found in red algae and in cryptomonad chloroplasts [START_REF] Bhattacharya | Genome of the red alga Porphyridium purpureum[END_REF][START_REF] Sturm | A novel type of light-harvesting antenna protein of red algal origin in algae with secondary plastids[END_REF]). The ochrophyte chloroplast additionally is defined by a « girdle lamella », a ring-like structure consisting of three apressed thylakoids that cover the exterior of the chloroplast stroma [START_REF] Andersen | Ultrastructure and 18S ribosomal RNA gene sequence for Pelagomonas calceolata gen. et sp. nov. and the description of a new algal class, the pelagophyceae classis nov[END_REF], and an additional synapomorphy (the accessory pigments diatoxanthin and diadinoxanthin) unifies the Khakista (diatoms and Bolidomonas) and hypogyristea (pelagophytes and dictyochophytes) [START_REF] Dorrell | Secondary plastids of stramenopiles[END_REF][START_REF] Buck | Lhcx proteins provide photoprotection via thermal dissipation of absorbed light in the diatom Phaeodactylum tricornutum[END_REF][START_REF] Kuczynska | Photosynthetic pigments in diatoms[END_REF]. (Figs. 1,2).

Cosnsistent with the classical paradigm based on biochemical and ultrastructural data, multigene phylogenies of chloroplast genomes robustly resolve ochrophytes as a monophyletic group, with the chloroplast originating within red algae, and closely related to other chloroplast groups acquired through the secondary endosymbiosis of red algae [START_REF] Dorrell | Chimeric origins of ochrophytes and haptophytes revealed through an ancient plastid proteome[END_REF][START_REF] Muñoz-Gómez | The new red algal subphylum proteorhodophytina comprises the largest and most divergent plastid genomes known[END_REF][START_REF] Stiller | The evolution of photosynthesis in chromist algae through serial endosymbioses[END_REF]. Thus, the most parsimonious scenario for the origin of the ochrophyte chloroplast is through a single endosymbiosis event in a recent common ancestor [START_REF] Dorrell | Chimeric origins of ochrophytes and haptophytes revealed through an ancient plastid proteome[END_REF], prior to the divergence of diatoms (Fig. 1).

Phylogenomic insights into diatom evolution

Prior to the genomics era, the deep evolutionary history of the eukaryotic tree was resolvable only through ultrastructural similarities, and single-gene trees realised with individual markers (e.g., 18S and chloroplast 16S ribosomal DNA [START_REF] Cavalier-Smith | A revised six-kingdom system of life[END_REF][START_REF] Cavalier-Smith | Principles of protein and lipid targeting in secondary symbiogenesis: euglenoid, dinoflagellate, and sporozoan plastid origins and the eukaryote family tree[END_REF]). Based on the conserved ultrastructural features of their chloroplasts, and single-gene phylogenetic data, it was posited that the major groups of algae with chloroplasts of secondary red origin, i.e., stramenopiles, cryptomonads, haptophytes, and dinoflagellates, descended from a common ancestor which acquired its chloroplast through a single secondary endosymbiosis involving a red alga [START_REF] Cavalier-Smith | A revised six-kingdom system of life[END_REF][START_REF] Cavalier-Smith | Principles of protein and lipid targeting in secondary symbiogenesis: euglenoid, dinoflagellate, and sporozoan plastid origins and the eukaryote family tree[END_REF]. These lineages were therefore proposed to form a monophyletic group, termed the "chromalveolates"; and non-photosynthetic members of these lineages, such as oomycetes, were posited to have therefore once possessed chloroplasts, and subsequently to have lost them [START_REF] Cavalier-Smith | A revised six-kingdom system of life[END_REF]. Whole genome sequencing in diatoms commenced with the centric species Thalassiosira pseudonana and the pennate Phaeodactylum tricornutum [START_REF] Armbrust | The genome of the diatom Thalassiosira pseudonana: ecology, evolution, and metabolism[END_REF][START_REF] Bowler | The Phaeodactylum genome reveals the evolutionary history of diatom genomes[END_REF]. This has also opened new windows of insight into the phylogenetic composition of the diatom nucleus [START_REF] Armbrust | The genome of the diatom Thalassiosira pseudonana: ecology, evolution, and metabolism[END_REF][START_REF] Bowler | The Phaeodactylum genome reveals the evolutionary history of diatom genomes[END_REF][START_REF] Moustafa | Genomic footprints of a cryptic plastid endosymbiosis in diatoms[END_REF], which has undergone substantial elaborations over its evolutionary history. The advent of highthroughput genomic sequencing, and therefore multigene phylogenomics revolutionised our understanding of diatom evolution, for example through the incorporation of rhizaria (which do not contain red algal chloroplasts) with stramenopiles and alveolates, as part of the "SAR clade" [START_REF] Burki | Phylogenomics reshuffles the eukaryotic supergroups[END_REF][START_REF] Walker | Eukaryotic systematics: a user's guide for cell biologists and parasitologists[END_REF]. Further studies have positioned the nuclear lineages of cryptomonads as being more closely related to that of plants and red algae than that of diatoms, directly questioning the occurrence of a single, secondary endosymbiosis event [START_REF] Burki | Untangling the early diversification of eukaryotes: a phylogenomic study of the evolutionary origins of Centrohelida, Haptophyta and Cryptista[END_REF][START_REF] Lax | Hemimastigophora is a novel supra-kingdom-level lineage of eukaryotes[END_REF]. Moreover, genomic investigation of major non-photosynthetic stramenopile groups, such as oomycetes [START_REF] Stiller | Are algal genes in nonphotosynthetic protists evidence of historical plastid endosymbioses?[END_REF] and hypochytriomycetes [START_REF] Leonard | Comparative genomic analysis of the 'pseudofungus' Hyphochytrium catenoides[END_REF], has revealed an absence of genes of red algal origin that are shared with photosynthetic lineages [START_REF] Stiller | The evolution of photosynthesis in chromist algae through serial endosymbioses[END_REF][START_REF] Wang | Re-analyses of "algal" genes suggest a complex evolutionary history of oomycetes[END_REF], placing the acquisition of the ochrophyte chloroplast after their divergence from non-photosynthetic relatives (Fig. 1).

The acquisition of a chloroplast by the common ancestor of the ochrophytes dramatically altered the composition of their genomes, adding a new organelle, with its own complex biochemical and physiological needs, to the host genome. Some of these functions are encoded in the chloroplast genome itself, and others in the genome of the stramenopile nucleus (Fig. 2). These nuclear genes, encoding chloroplast-targeted proteins, might have originated within the nucleus itself, and adapted to support the biology of the chloroplast (59-61), or might have been derived from either the nucleus or the chloroplast of the red algal ancestor of the ochrophyte chloroplast, and relocated to the host via endosymbiotic gene transfer (2, 39) (Fig. 2). At least some of the proteins in the chloroplast proteome may additionally have bifunctional roles in other organelles, e.g. through dual-targeting to the mitochondria [START_REF] Dorrell | Principles of plastid reductive evolution illuminated by nonphotosynthetic chrysophytes[END_REF][START_REF] Dorrell | Chimeric origins of ochrophytes and haptophytes revealed through an ancient plastid proteome[END_REF][START_REF] Gile | Dual organellar targeting of aminoacyl-tRNA synthetases in diatoms and cryptophytes[END_REF] or to the plasma membrane (63) (Fig. 2).

Alongside this, both the nuclear and mitochondrial genomes of ochrophytes and aplastidic stramenopiles have undergone their own, independent gene transfer events, e.g., the relocation of mitochondria-encoded functions to the nucleus, and the horizontal acquisition of nonchloroplast related genes of non-stramenopile origin by the stramenopile nuclear genome [START_REF] Bowler | The Phaeodactylum genome reveals the evolutionary history of diatom genomes[END_REF][START_REF] Keeling | Horizontal gene transfer in eukaryotic evolution[END_REF][START_REF] Rastogi | Integrative analysis of large scale transcriptome data draws a comprehensive landscape of Phaeodactylum tricornutum genome and evolutionary origin of diatoms[END_REF]. Here, we summarise current knowledge (as of 2021) regarding different evolutionary sources of novelty in diatom nuclear genomes that have arisen through the course of stramenopile evolution, focusing on the genome of the model diatom P. tricornutum, which remains the best-studied system for phylogenomic reconstructions of gene transfer in the diatom lineage [START_REF] Bowler | The Phaeodactylum genome reveals the evolutionary history of diatom genomes[END_REF][START_REF] Rastogi | Integrative analysis of large scale transcriptome data draws a comprehensive landscape of Phaeodactylum tricornutum genome and evolutionary origin of diatoms[END_REF] (Table 1). Subsequently, we explore the dynamic evolution of diatom mitochondria and chloroplasts, and identify features underpinning the progressive loss, and occasional gain, of novel coding functions in the organelle genomes of individual diatom species.

Methods and problems when exploring origins of diatom nuclear genes

Dynamic transitions in the evolutionary history of the diatom nuclear, mitochondrial, and chloroplast genomes can be identified, and quantified, by comparative genomic and phylogenomic approaches [START_REF] Bowler | The Phaeodactylum genome reveals the evolutionary history of diatom genomes[END_REF][START_REF] Moustafa | Genomic footprints of a cryptic plastid endosymbiosis in diatoms[END_REF][START_REF] Rastogi | Integrative analysis of large scale transcriptome data draws a comprehensive landscape of Phaeodactylum tricornutum genome and evolutionary origin of diatoms[END_REF] (Table 1). Understanding these transitions is important as it may reveal adaptations of diatoms to their current environments, and allow us to pinpoint genomic novelties that may explain their success over red or green algae, or even other stramenopile groups, in the contemporary ocean [START_REF] Dorrell | Do red and green make brown?: Perspectives on plastid acquisitions within chromalveolates[END_REF]. However, the exact size, timing, reason, and even the veracity, of individual gene transfer events can be controversial [START_REF] Deschamps | Re-evaluating the green contribution to diatom genomes[END_REF][START_REF] Dagan | Seeing green and red in diatom genomes[END_REF], depending particularly on the methodology used.

Firstly, different results may be obtained dependent on the reference library used, which may be constrained both by taxonomic undersampling and oversampling of key lineages. For example, early estimates of the green algal signal in diatom genomes [START_REF] Armbrust | The genome of the diatom Thalassiosira pseudonana: ecology, evolution, and metabolism[END_REF][START_REF] Bowler | The Phaeodactylum genome reveals the evolutionary history of diatom genomes[END_REF][START_REF] Ponce-Toledo | Horizontal and endosymbiotic gene transfer in early plastid evolution[END_REF] were likely to be overinflated by the paucity of red algal gene models at the time of analysis, being largely dependent on the highly reduced genome of the extremophilic alga Cyanidioschyzon merolae as the only publicly available red algal gene reference [START_REF] Matsuzaki | Genome sequence of the ultrasmall unicellular red alga Cyanidioschyzon merolae 10D[END_REF][START_REF] Collén | Genome structure and metabolic features in the red seaweed Chondrus crispus shed light on evolution of the Archaeplastida[END_REF].

In contrast, the indiscriminate use of transcriptome data and poorly curated reference genomes can lead to the misidentification of contaminants as horizontally transferred genes [START_REF] Dorrell | Chimeric origins of ochrophytes and haptophytes revealed through an ancient plastid proteome[END_REF][START_REF] Vancaester | Comprehensive and functional analysis of horizontal gene transfer events in diatoms[END_REF][START_REF] Burki | Re-evaluating the green versus red signal in eukaryotes with secondary plastid of red algal origin[END_REF], or the preferential identification of genes with unresolvable or ambiguous origins as being related to oversampled taxa [START_REF] Deschamps | Re-evaluating the green contribution to diatom genomes[END_REF]. These strategies may be avoided by judicious taxonomic sampling when constructing reference datasets [START_REF] Dorrell | Chimeric origins of ochrophytes and haptophytes revealed through an ancient plastid proteome[END_REF][START_REF] Morozov | Diatom genes originating from red and green algae: Implications for the secondary endosymbiosis models[END_REF]; alongside the use of new long-read sequencing technologies (e.g. ; PacBio (74)), assembly approaches (e.g.,; Dovetail (75)), gene annotation pipelines (incorporative of transcriptome or proteomic data (65, 76)), and in silico cleaning of reference genome and transcriptome libraries (e.g., through considering nucleotide composition, [START_REF] Dorrell | Phylogenomic fingerprinting of tempo and functions of horizontal gene transfer within ochrophytes[END_REF][START_REF] Hehenberger | Functional relationship between a dinoflagellate host and its diatom endosymbiont[END_REF][START_REF] Sato | Genome-enabled phylogenetic and functional reconstruction of an araphid pennate diatom Plagiostriata sp. CCMP470, previously assigned as a radial centric diatom, and its bacterial commensal[END_REF]) to improve curation of gene models, and remove probable contaminant sequences.

Different results can also be obtained because of the methodology used, e.g., BLAST-based analyses such as ranking of top hits [START_REF] Stiller | The evolution of photosynthesis in chromist algae through serial endosymbioses[END_REF][START_REF] Armbrust | The genome of the diatom Thalassiosira pseudonana: ecology, evolution, and metabolism[END_REF][START_REF] Rastogi | Integrative analysis of large scale transcriptome data draws a comprehensive landscape of Phaeodactylum tricornutum genome and evolutionary origin of diatoms[END_REF][START_REF] Méheust | Protein networks identify novel symbiogenetic genes resulting from plastid endosymbiosis[END_REF] via whole-genome phylogenomic analysis [START_REF] Dorrell | Phylogenomic fingerprinting of tempo and functions of horizontal gene transfer within ochrophytes[END_REF][START_REF] Morozov | Diatom genes originating from red and green algae: Implications for the secondary endosymbiosis models[END_REF][START_REF] Deschamps | Re-evaluating the green contribution to diatom genomes[END_REF][START_REF] Vancaester | Comprehensive and functional analysis of horizontal gene transfer events in diatoms[END_REF][START_REF] Fan | Phytoplankton pangenome reveals extensive prokaryotic horizontal gene transfer of diverse functions[END_REF]. BLAST top hit analysis is less computationally intensive than phylogenetic techniques, and provides several technical advantages, e.g., the ability to infer possible evolutionary histories for genes that are too short or divergently evolved (e.g., chimeric gene fusions) to be resolved through classical phylogenetic approaches [START_REF] Dorrell | Chimeric origins of ochrophytes and haptophytes revealed through an ancient plastid proteome[END_REF][START_REF] Dorrell | Phylogenomic fingerprinting of tempo and functions of horizontal gene transfer within ochrophytes[END_REF][START_REF] Méheust | Protein networks identify novel symbiogenetic genes resulting from plastid endosymbiosis[END_REF], but its resolution is necessarily poorer, for example not being able to determine the directions of gene transfers that can be inferred from the topology (e.g., monophyly versus paraphyly of stramenopile lineages) within a phylogenetic output [START_REF] Dorrell | Chimeric origins of ochrophytes and haptophytes revealed through an ancient plastid proteome[END_REF][START_REF] Morozov | Diatom genes originating from red and green algae: Implications for the secondary endosymbiosis models[END_REF].

The threshold stringencies applied when identifying evolutionary origins may also bias the evolutionary relationships reconstructed. Too permissive criteria can admit poorly resolved or artifactual relationships, whereas too stringent parameters can conversely eliminate usable phylogenetic signals. For example, using phylogenomic approaches and automated tree sorting, Moustafa et al identified 418 red and 1,757 green genes in the P. tricornutum genome (56) (Table 1). Following manual reinspection of the green gene dataset identified by Moustafa et al [START_REF] Moustafa | Genomic footprints of a cryptic plastid endosymbiosis in diatoms[END_REF], Deschamps and Moreira found that 91 of the putative green gene trees had topologies consistent with a vertical transfer of genes from the green lineage into the diatoms, whereas 89 of the putative green genes had histories more consistent with a transfer from the red lineage into the diatoms (66), although they did not perform a comparable analysis of the red genes identified by Moustafa et al. (Table 1).

Finally, even within well-sampled datasets, the exact questions that are posed by a study of horizontal gene transfer will determine the results obtained. Early studies on diatoms necessarily focussed on gene transfers from animals, fungi, bacteria, red algae and green algae, as these were the reference genomic models available at the time [START_REF] Armbrust | The genome of the diatom Thalassiosira pseudonana: ecology, evolution, and metabolism[END_REF][START_REF] Bowler | The Phaeodactylum genome reveals the evolutionary history of diatom genomes[END_REF][START_REF] Moustafa | Genomic footprints of a cryptic plastid endosymbiosis in diatoms[END_REF]. However, diatoms may also have exchanged genes with other algae with secondary chloroplasts (e.g., cryptomonads, haptophytes and dinoflagellates); either as part of chloroplast endosymbiosis events [START_REF] Dorrell | Chimeric origins of ochrophytes and haptophytes revealed through an ancient plastid proteome[END_REF][START_REF] Dorrell | Phylogenomic fingerprinting of tempo and functions of horizontal gene transfer within ochrophytes[END_REF][START_REF] Stiller | The evolution of photosynthesis in chromist algae through serial endosymbioses[END_REF] or through independent horizontal gene transfers [START_REF] Nonoyama | Metabolic Innovations Underpinning the Origin and Diversification of the Diatom Chloroplast[END_REF][START_REF] Kazamia | Endocytosis-mediated siderophore uptake as a strategy for Fe acquisition in diatoms[END_REF]; and some of the "bacterial", "red" or "green" genes identified in early studies may have passed through one or more of these algal groups as intermediates prior to arriving in diatom genomes [START_REF] Dorrell | Phylogenomic fingerprinting of tempo and functions of horizontal gene transfer within ochrophytes[END_REF]. Genes acquired horizontally into diatom genomes may have variously been received in early ancestors of the SAR clade, stramenopiles or ochrophytes, in the diatom ancestor, or even specifically within the diatom species considered; and recent studies have profited from the expanded number of diatom and stramenopile genomes and transcriptomes available to infer the probable timing of individual gene transfers [START_REF] Dorrell | Phylogenomic fingerprinting of tempo and functions of horizontal gene transfer within ochrophytes[END_REF][START_REF] Vancaester | Comprehensive and functional analysis of horizontal gene transfer events in diatoms[END_REF][START_REF] Fan | Phytoplankton pangenome reveals extensive prokaryotic horizontal gene transfer of diverse functions[END_REF]. These more densely sampled datasets may even be able to identify occasions in which diatoms or ancestors have acted as donors, rather than recipients, in gene transfers with other lineages [START_REF] Dorrell | Chimeric origins of ochrophytes and haptophytes revealed through an ancient plastid proteome[END_REF][START_REF] Dorrell | Phylogenomic fingerprinting of tempo and functions of horizontal gene transfer within ochrophytes[END_REF].

In Table 1, we profile the key results of different studies of horizontal gene transfers involving the diatom nucleus, focussing on the T. pseudonana and P. tricornutum genomes [START_REF] Dorrell | Chimeric origins of ochrophytes and haptophytes revealed through an ancient plastid proteome[END_REF][START_REF] Dorrell | Phylogenomic fingerprinting of tempo and functions of horizontal gene transfer within ochrophytes[END_REF][START_REF] Armbrust | The genome of the diatom Thalassiosira pseudonana: ecology, evolution, and metabolism[END_REF][START_REF] Bowler | The Phaeodactylum genome reveals the evolutionary history of diatom genomes[END_REF][START_REF] Moustafa | Genomic footprints of a cryptic plastid endosymbiosis in diatoms[END_REF][START_REF] Rastogi | Integrative analysis of large scale transcriptome data draws a comprehensive landscape of Phaeodactylum tricornutum genome and evolutionary origin of diatoms[END_REF][START_REF] Deschamps | Re-evaluating the green contribution to diatom genomes[END_REF][START_REF] Vancaester | Comprehensive and functional analysis of horizontal gene transfer events in diatoms[END_REF][START_REF] Fan | Phytoplankton pangenome reveals extensive prokaryotic horizontal gene transfer of diverse functions[END_REF]. The most recent of these analyses, involving a densely sampled reference dataset of all currently available eukaryotic and prokaryotic genomes and transcriptomes, and manual sorting of trees by partner lineage, timing and direction of horizontal transfer [START_REF] Dorrell | Phylogenomic fingerprinting of tempo and functions of horizontal gene transfer within ochrophytes[END_REF]; identified 1,347/ 12,177 (11.0%) genes in the P. tricornutum genome to have been horizontally acquired since the origin of the ochrophytes, and a further 1,771 examples, distributed over 1,184 (9.7%) genes, of gene transfers from the ochrophytes into the other branches of the tree of life [START_REF] Dorrell | Phylogenomic fingerprinting of tempo and functions of horizontal gene transfer within ochrophytes[END_REF]. Below, we discuss the likely origin points and functions of different horizontally transferred genes, including genes acquired from bacteria, red algae and their endosymbiotic descendants, green algae, as well as gene transfers from diatoms into other eukaryotic lineages.

Prokaryotic signals

Phylogenomic annotations of the P. tricornutum have identified large numbers of genes of prokaryotic origin, inferred to constitute 7.4% (784 genes) of the original genome annotation [START_REF] Bowler | The Phaeodactylum genome reveals the evolutionary history of diatom genomes[END_REF]; and reduced to a still substantial 285 genes (2.3%) [START_REF] Dorrell | Phylogenomic fingerprinting of tempo and functions of horizontal gene transfer within ochrophytes[END_REF] in the most recent estimates from the third genome annotation with more densely sampled taxonomic references (Table 1) [START_REF] Dorrell | Phylogenomic fingerprinting of tempo and functions of horizontal gene transfer within ochrophytes[END_REF]. Most of these genes pertain to bacteria, with limited or zero estimated archaeal and viral contributions into diatom genomes [START_REF] Dorrell | Phylogenomic fingerprinting of tempo and functions of horizontal gene transfer within ochrophytes[END_REF][START_REF] Rastogi | Integrative analysis of large scale transcriptome data draws a comprehensive landscape of Phaeodactylum tricornutum genome and evolutionary origin of diatoms[END_REF]. These genes have been acquired progressively through diatom evolution, with both studies focussed exclusively on Phaeodactylum [START_REF] Dorrell | Phylogenomic fingerprinting of tempo and functions of horizontal gene transfer within ochrophytes[END_REF][START_REF] Rastogi | Integrative analysis of large scale transcriptome data draws a comprehensive landscape of Phaeodactylum tricornutum genome and evolutionary origin of diatoms[END_REF]; and indeed on wider diatom pan-genomes [START_REF] Dorrell | Phylogenomic fingerprinting of tempo and functions of horizontal gene transfer within ochrophytes[END_REF][START_REF] Vancaester | Comprehensive and functional analysis of horizontal gene transfer events in diatoms[END_REF][START_REF] Fan | Phytoplankton pangenome reveals extensive prokaryotic horizontal gene transfer of diverse functions[END_REF], detecting large numbers of bacterially derived genes into early ancestors of the SAR clade, stramenopiles or ochrophytes; into the diatom common ancestor; and even into individual diatom groups or species (Table 1).

Two recent studies from Vancaester et al. (2020) (67) and from our group [START_REF] Dorrell | Phylogenomic fingerprinting of tempo and functions of horizontal gene transfer within ochrophytes[END_REF], which have reconciled automatically resolved bacterial transfers from densely sampled datasets with multigene reference tree topologies for SAR clade members, have attributed large numbers of bacterial gene transfer to early ancestors of the diatoms, following their divergence from other stramenopiles (40, 67) (Table 1). This may reflect a greater flux of bacterial DNA into diatoms than related algae, which may contribute to their comparative ecological success [START_REF] Dorrell | Phylogenomic fingerprinting of tempo and functions of horizontal gene transfer within ochrophytes[END_REF]. However, it is possible that this asymmetry reflects the greater number of diatom reference sequence libraries in which gene transfers may be identified, and a parallel study by Fan et al.

(2020) using a smaller number of diatom genomes, but balanced taxonomic sampling of diatom and non-diatom species, did not appear to yield greater number of bacterial genes in diatoms than other lineages (68) (Table 1). An alternative explanation is a high rate of transfer, but also secondary loss of bacterial-derived genes into diatoms, which may enable specific diatom species to explore new evolutionary niches and fluctuating environments [START_REF] Dorrell | Phylogenomic fingerprinting of tempo and functions of horizontal gene transfer within ochrophytes[END_REF].

Regardless of the frequencies of their arrival, bacterial genes contribute a wide range of different functions to diatoms. For example, within the dataset of 770 genes encoding chloroplast-targeted proteins that are shared across (and presumably ancestral to) all ochrophytes, we identified 49 of probable prokaryotic origin, including many implicated in the expression of the chloroplast genome [START_REF] Dorrell | Chimeric origins of ochrophytes and haptophytes revealed through an ancient plastid proteome[END_REF]. More recently, considering both in silico targeting predictions and GFP labelling, we have shown that bacterial genes acquired early during ochrophyte evolution are enriched in chloroplast-targeted proteins, consistent with origins tied to the ochrophyte chloroplast; whereas more recently acquired proteins predominantly function in the diatom secretome [START_REF] Dorrell | Phylogenomic fingerprinting of tempo and functions of horizontal gene transfer within ochrophytes[END_REF]. Many of these more recently acquired bacterial genes have functions pertaining to environmental stress, e.g., ice-binding proteins in polar-adapted diatoms [START_REF] Sorhannus | Evolution of antifreeze protein genes in the diatom genus fragilariopsis: evidence for horizontal gene transfer, gene duplication and episodic diversifying selection[END_REF][START_REF] Raymond | Possible role of horizontal gene transfer in the colonization of sea ice by algae[END_REF]; and proteins involved in the synthesis of Vitamin B 12 , which may be a limiting micronutrient in many of the Southern Ocean habitats where diatoms are abundant [START_REF] Vancaester | Comprehensive and functional analysis of horizontal gene transfer events in diatoms[END_REF][START_REF] Browning | Nutrient co-limitation at the boundary of an oceanic gyre[END_REF].

Red algal-related signals

Early estimates posited approximately 400 genes (4% of the coding content )in the P. tricornutum and T. pseudonana genomes were of red algal origin (Table 1) [START_REF] Bowler | The Phaeodactylum genome reveals the evolutionary history of diatom genomes[END_REF][START_REF] Moustafa | Genomic footprints of a cryptic plastid endosymbiosis in diatoms[END_REF][START_REF] Rastogi | Integrative analysis of large scale transcriptome data draws a comprehensive landscape of Phaeodactylum tricornutum genome and evolutionary origin of diatoms[END_REF]. These genes principally encode proteins of chloroplast function, and are shared across ochrophytes, e.g., constituting one half (255 of the 502 genes) of the chloroplast-targeted proteins shared across all ochrophytes identified by Dorrell et al. 2017 for which a tractable phylogenetic signal could be found (Table 1) [START_REF] Dorrell | Chimeric origins of ochrophytes and haptophytes revealed through an ancient plastid proteome[END_REF]. In contrast, only four of these genes were found to be shared with non-photosynthetic stramenopile groups, e.g., in oomycetes and labyrinthulomycetes (39) (Table 1). This, alongside the previously discussed lack of red algal signal in the genomes of non-photosynthetic stramenopiles [START_REF] Stiller | Are algal genes in nonphotosynthetic protists evidence of historical plastid endosymbioses?[END_REF][START_REF] Leonard | Comparative genomic analysis of the 'pseudofungus' Hyphochytrium catenoides[END_REF][START_REF] Stiller | The evolution of photosynthesis in chromist algae through serial endosymbioses[END_REF][START_REF] Wang | Re-analyses of "algal" genes suggest a complex evolutionary history of oomycetes[END_REF] is consistent with a late acquisition of the ochrophyte chloroplast, after their divergence from oomycetes (2).

Despite the massive expansion in the availability of red algal genome sequences and transcriptomes in the last decade [START_REF] Dorrell | Chimeric origins of ochrophytes and haptophytes revealed through an ancient plastid proteome[END_REF][START_REF] Collén | Genome structure and metabolic features in the red seaweed Chondrus crispus shed light on evolution of the Archaeplastida[END_REF][START_REF] Brawley | Insights into the red algae and eukaryotic evolution from the genome of Porphyra umbilicalis (Bangiophyceae, Rhodophyta)[END_REF], the number of red algal-derived genes identified in stramenopile genomes has remained relatively constant. For example, whereas Moustafa et al. ( 2009) identified 418 genes of putative red origin in the P. tricornutum genome using the extremophilic red alga C. merolae as a principle reference (56), only 459 red genes were identified by Rastogi et al. (2018) using a much more exhaustive dataset including five complete red algal genomes, and thirteen different red algal transcriptomes (65) (Table 1). One possible reason for this quite limited red contribution is the relatively high level of reduction observed in red algal genomes, which lack many of the accessory functions identified in diatoms and in other eukaryotic groups [START_REF] Rastogi | Integrative analysis of large scale transcriptome data draws a comprehensive landscape of Phaeodactylum tricornutum genome and evolutionary origin of diatoms[END_REF][START_REF] Qiu | Hypothesis: Gene-rich plastid genomes in red algae may be an outcome of nuclear genome reduction[END_REF].

An alternative scenario is that the red algal signal in stramenopile lineages does not itself directly come from red algae, but from a secondary, red-chloroplast containing lineage that was acquired by the stramenopiles through a tertiary or more complex endosymbiosis event.

Using an innovative methodology based on the similarity of BLAST hit signals, Stiller et al.

(2014) concluded a probable cryptomonad origin for the ochrophyte chloroplast [START_REF] Stiller | The evolution of photosynthesis in chromist algae through serial endosymbioses[END_REF]. In our most recent analyses, considering algae with secondary chloroplasts as possible donors into diatom genomes, we could only manually identify 193 P. tricornutum genes that resolved phylogenetically as a direct sister to red algae, but found a further 514 that resolved with cryptomonads, haptophytes or dinoflagellates, with haptophytes contributing the greatest number (Table 1) [START_REF] Dorrell | Phylogenomic fingerprinting of tempo and functions of horizontal gene transfer within ochrophytes[END_REF]. In this case, whatever functions that might have been inherited by the stramenopile ancestor from red algae would have been winnowed by not only one, but potentially two or more successive endosymbiotic events. Ultimately, identifying why stramenopiles contain the red algal genes that they do will depend on tracing the permeation of the red signal across all major groups of algae with secondary, red-derived chloroplasts.

Green algal-derived signals

As discussed above, diatoms contain a sizeable number of genes of green algal origin, although the number identified varies based on the methodology employed (56, 61, 66) (Table 1). In our most recent estimates, we identified 260 (2%) genes in the P. tricornutum genome showing relationships consistent with a direct gene transfer from the green algae into the ochrophytes, most of which resolve to deep nodes in ochrophyte evolution (Table 1) [START_REF] Dorrell | Phylogenomic fingerprinting of tempo and functions of horizontal gene transfer within ochrophytes[END_REF], and 105 out of 502 (20%) of conserved ochrophyte chloroplast-targeted proteins for which a tractable origin was identified for green algae (39) (Table 1). The exact numbers and identities of the green genes in diatom genomes is likely to undergo further revision with deeper sequencing of red algae (e.g., xanthophyll cycle genes ( 85)), and the reassignation of some putatively "green" genes as having cryptomonad, haptophyte or dinoflagellate origins; although even the most stringent pipelines and sampling thresholds fail to completely eliminate the green signal from diatom genomes [START_REF] Dorrell | Phylogenomic fingerprinting of tempo and functions of horizontal gene transfer within ochrophytes[END_REF][START_REF] Deschamps | Re-evaluating the green contribution to diatom genomes[END_REF].

Beyond the presence and size of the green signal in diatoms, other questions remain, namely: where do these genes come from, and how were they acquired? Phylogenomic analyses typically pinpoint green genes as arising within chlorophytes (i.e., single-celled green algae) rather than streptophytes (plants and their closest relatives), which helps argue against their origin as being artifactual because of phylogenetic misannotation [START_REF] Dorrell | Chimeric origins of ochrophytes and haptophytes revealed through an ancient plastid proteome[END_REF][START_REF] Moustafa | Genomic footprints of a cryptic plastid endosymbiosis in diatoms[END_REF], although it remains to be determined where within the chlorophytes this signal preferentially falls (Table 1). The green genes that are present in diatom genomes preferentially encode chloroplast-targeted proteins [START_REF] Dorrell | Chimeric origins of ochrophytes and haptophytes revealed through an ancient plastid proteome[END_REF], which might be consistent with a chloroplast endosymbiotic origin. This could be reconcilable either through a cryptic green endosymbiosis within the evolutionary history of the stramenopile host, or through the acquisition of a tertiary or higher chloroplast containing mixed signals of red and green origin (2, 5). Answering precisely how green genes were transferred into diatoms will depend both on deeper sequencing of the green algal tree (particularly in the context of early-diverging members, e.g. Prasinoderma ( 86)); and ideally the verification of the exact evolutionary origin of the ochrophyte chloroplasts (from red algae, cryptomonads, or another group entirely) [START_REF] Stiller | The evolution of photosynthesis in chromist algae through serial endosymbioses[END_REF].

Perhaps most interesting is to consider what functional advantages green genes might contribute to diatom genomes. Many of the green genes encoding chloroplast-targeted proteins have functions in biosynthetic pathways (e.g., chloroplast and carotenoid synthesis (2, 87, 88)). Other diatom green genes have functions in both chloroplast-and nonchloroplast related environmental stress responses, e.g., including Lhcx light-harvesting complex genes implicated in photoadaptation to aberrant light conditions [START_REF] Buck | Lhcx proteins provide photoprotection via thermal dissipation of absorbed light in the diatom Phaeodactylum tricornutum[END_REF][START_REF] Büchel | Evolution and function of light harvesting proteins[END_REF], and the iron-stress related protein ISIP2a, which is mediates non-reductive iron uptake across the plasma membrane [START_REF] Mcquaid | Carbonate-sensitive phytotransferrin controls high-affinity iron uptake in diatoms[END_REF][START_REF] Allen | Whole-cell response of the pennate diatom Phaeodactylum tricornutum to iron starvation[END_REF]. Understanding the functional significance of the green footprint in diatom genomes will depend on large scale characterisation of their encoded properties, e.g., using environmental sequence datasets, and targeted mutagenesis in model taxa [START_REF] Dorrell | Do red and green make brown?: Perspectives on plastid acquisitions within chromalveolates[END_REF].

Have diatoms donated genes to other organisms ?

Alongside the mosaic origin of diatom genomes, it is possible that diatoms, or ochrophytes in general, have donated genes to other organisms. For example, we previously identified 243 conserved chloroplast-targeted proteins that have been shared between the ochrophytes and the haptophytes [START_REF] Dorrell | Chimeric origins of ochrophytes and haptophytes revealed through an ancient plastid proteome[END_REF]; which we have subsequently expanded to 817 genes (6.7%) of the Phaeodactylum genome supporting horizontal gene transfer from ochrophytes to haptophytes (40) (Table 1). Phylogenetic analysis of these genes indicated a mass transfer event from the hypogyristea (pelagophytes and dictyochophytes) into a common haptophyte ancestor [START_REF] Dorrell | Secondary plastids of stramenopiles[END_REF][START_REF] Dorrell | Chimeric origins of ochrophytes and haptophytes revealed through an ancient plastid proteome[END_REF][START_REF] Dorrell | Phylogenomic fingerprinting of tempo and functions of horizontal gene transfer within ochrophytes[END_REF]. These genes predominantly have chloroplast-targeted functions, and may be footprints of an ancient endosymbiotic transfer of the ochrophyte chloroplast into the haptophytes [START_REF] Dorrell | Chimeric origins of ochrophytes and haptophytes revealed through an ancient plastid proteome[END_REF][START_REF] Stiller | The evolution of photosynthesis in chromist algae through serial endosymbioses[END_REF].

We have subsequently inferred the same enrichment in pelagophyte/ dictyochophyte affinities for genes transferred from the ochrophytes to the dinoflagellates, which may relate to a direct or indirect (e.g., via haptophyte) gene transfer (40) (Table 1). Further phylogenetic analysis, of the chloroplast genomes of the photosynthetic alveolates Chromera and Vitrella has revealed a possible relationship with chrysophytes in the ochrophytes [START_REF] Ševčíková | Updating algal evolutionary relationships through plastid genome sequencing: did alveolate plastids emerge through endosymbiosis of an ochrophyte?[END_REF][START_REF] Dorrell | Phylogenomic fingerprinting of tempo and functions of horizontal gene transfer within ochrophytes[END_REF][START_REF] Kim | Evolutionary dynamics of cryptophyte plastid genomes[END_REF]. These serial transfer events may help resolve the still uncertain origins, dynamics and functions of the red and green algal signals associated with marine algal genomes.

Despite the gene transfers associated with other ochrophytes, there is relatively little evidence that diatoms themselves have transferred large numbers of genes into other algal groups-with one exception. The « dinotoms », a closely related group of dinoflagellate algae within the order Peridiniales, possess whole cell endosymbionts of diatom origin [START_REF] Imanian | The complete plastid genomes of the two 'dinotoms' Durinskia baltica and Kryptoperidinium foliaceum[END_REF][START_REF] Yamada | Discovery of a kleptoplastic 'dinotom' dinoflagellate and the unique nuclear dynamics of converting kleptoplastids to permanent plastids[END_REF]. These endosymbionts, which retain a complete chloroplast, mitochondria, and nucleus, appear to have been acquired, lost and replaced on multiple occasions, from both pennate and centric diatom lineages [START_REF] Kretschmann | Molecular phylogenetics of dinophytes harboring diatoms as endosymbionts (Kryptoperidiniaceae, Peridiniales), with evolutionary interpretations and a focus on the identity of Durinskia oculata from Prague[END_REF][START_REF] Yamada | Identification of highly divergent diatomderived chloroplasts in dinoflagellates, including a description of Durinskia kwazulunatalensis sp. nov. (Peridiniales, Dinophyceae)[END_REF][START_REF] Yamada | Five non-motile dinotom dinoflagellates of the genus Dinothrix[END_REF]. Phylogenomic analysis of dinotom nuclear transcriptomes reveals very little evidence for the loss of functions from the symbionts, or transfer of genes to the host, consistent with their relatively transient evolutionary associations [START_REF] Hehenberger | Functional relationship between a dinoflagellate host and its diatom endosymbiont[END_REF][START_REF] Burki | Endosymbiotic gene transfer in tertiary plastid-containing dinoflagellates[END_REF]. It remains to be determined what genetic integration events, if any, are required for the stable domestication of the dinotom endosymbionts.

Despite their relatively limited evolutionary interactions with other algal groups, diatoms may exchange genes with each other, which may have adaptive functions to certain environmental stresses. For example, the iron stress-associated protein ISIP1, which facilitates the nonreductive uptake of extracellular siderophores by endocytosis, shows a complex phylogeny, consistent with either multiple transfer events between diatoms, or independent paralogy and gene loss events [START_REF] Kazamia | Endocytosis-mediated siderophore uptake as a strategy for Fe acquisition in diatoms[END_REF]. Similarly, phylogenies of ice-binding proteins, which confer cold stress tolerance in polar native algae, reveal probable horizontal transfer events between centric and pennate species (e.g., between the centric diatom Chaetoceros and the pennate Navicula sp.; or between the centric Attheya and the pennate Amphora sp. [START_REF] Sorhannus | Evolution of antifreeze protein genes in the diatom genus fragilariopsis: evidence for horizontal gene transfer, gene duplication and episodic diversifying selection[END_REF][START_REF] Raymond | Possible role of horizontal gene transfer in the colonization of sea ice by algae[END_REF]). Resolving these more recent gene transfer events is more challenging, as they are more likely to be biased by limitations in taxonomic sampling, and limited resolution of short protein sequences [START_REF] Swenson | Finding a partner in the ocean: molecular and evolutionary bases of the response to sexual cues in a planktonic diatom[END_REF]. However, explorations of adaptive and within-genus diatom gene transfers is becoming increasingly possible due to dense transcriptomic sampling of specific genera (e.g., Thalassiosira, Chaetoceros ; Fig. 1 (6)) or genomes (Thalassiosira, Pseudo-nitzschia ; (100)).

Evolution of the diatom organelle genomes

Diatom mitochondria

Diatom mitochondrial genomes display a largely well-conserved organisation, forming a single, circular chromosome with a single repeat region [START_REF] Kamikawa | Diversity of organellar genomes in non-photosynthetic diatoms[END_REF]101). These genomes typically contain 33 core protein-coding genes, alongside genes for tRNAs and rRNAs (Fig. 3A) (101-103). This coding content is less than in some microbial eukaryotes (e.g., 47 protein-coding genes in the free-living stramenopile relative Ancoracystis), but somewhat greater than the three protein-coding genes retained in dinoflagellate mitochondria (104, 105). The coding content of diatom mitochondria is similar to that of both ochrophytes and non-photosynthetic stramenopiles (Fig. 4A), except for the probable ancestral loss of atp1 in diatoms, which is mitochondria-encoded in both photosynthetic eustigmatophytes (106) and non-photosynthetic oomycetes and bicosoecids [START_REF] Jirsová | Morphology, ultrastructure, and mitochondrial genome of the marine non-photosynthetic bicosoecid Cafileria marina gen. et sp[END_REF]. A further seven genes have more sporadic distributions across diatom mitochondria, including tatA, which is only mitochondria-encoded in a few select genera (13, 101, 107) (Fig. 3B).

Alongside this relatively conserved genomic content, more unusual organisations have evolved in individual mitochondrial genomes. These include structural changes, e.g., a possible linear organisation of the mitochondrial genome in the secondarily nonphotosynthetic diatom Nitzschia [START_REF] Kamikawa | Diversity of organellar genomes in non-photosynthetic diatoms[END_REF] iron-sulfur binding and the molybdopterin-binding domains (103, 108, 110). This configuration is not known in araphid pennate or centric diatom mitochondria, and its functional consequences remain unknown (110).

Diatom chloroplast genomes

The chloroplast genomes associated with diatoms are typically arranged as a single, circular chromosome, with between 134 and 180 protein-coding genes, alongside ribosomal and transfer RNAs (Fig. 4) [START_REF] Dorrell | Secondary plastids of stramenopiles[END_REF][START_REF] Yu | Evolution of the plastid genomes in diatoms[END_REF]111,112). Phylogenomic analysis, and the presence of discrete molecular synapomorphies (e.g., the presence of a form ID type rubisco) robustly place this chloroplast genome within red algae, and closely related to the chloroplasts of cryptomonads and haptophytes [START_REF] Muñoz-Gómez | The new red algal subphylum proteorhodophytina comprises the largest and most divergent plastid genomes known[END_REF]113,114). However, the coding content of the diatom chloroplast genome is somewhat less than the ca. 250 genes associated with red algal chloroplasts [START_REF] Muñoz-Gómez | The new red algal subphylum proteorhodophytina comprises the largest and most divergent plastid genomes known[END_REF][START_REF] Qiu | Hypothesis: Gene-rich plastid genomes in red algae may be an outcome of nuclear genome reduction[END_REF], and indeed is less than the chloroplast gene contents of other ochrophyte groups ( 2), e.g., raphidophytes (Heterosigma) [START_REF] Cattolico | Chloroplast genome sequencing analysis of Heterosigma akashiwo CCMP452 (West Atlantic) and NIES293 (West Pacific) strains[END_REF], phaeophytes (Ectocarpus) [START_REF] Corguillé | Plastid genomes of two brown algae, Ectocarpus siliculosus and Fucus vesiculosus: further insights on the evolution of red-algal derived plastids[END_REF] and chrysophytes (Ochromonas) [START_REF] Ševčíková | Updating algal evolutionary relationships through plastid genome sequencing: did alveolate plastids emerge through endosymbiosis of an ochrophyte?[END_REF], pointing to both endosymbiotic-and post-endosymbiotic reductions in diatom chloroplast genome content (Fig. 3). This notwithstanding, the diatom chloroplast genome has undergone less dramatic reductions than some other ochrophyte groups (e.g., pelagophytes, dictyochophytes), in which even the loss of the chloroplast inverted repeat is known [START_REF] Han | Dictyochophyceae plastid genomes reveal unusual variability in their organization[END_REF][START_REF] Ong | Analyses of the commplete chloroplast genome sequences of two members of the Pelagophyceae: Aureococcus anophageferrens CCMP1984 and Aureooumbra lagunensis CCMP1507[END_REF].

Alongside these general trends, different diatom species have retained and lost different patterns of genes (Fig. 5). For example, the basally divergent genus Leptocylindrus (4) retains a chloroplast petJ gene encoding cytochrome c § , which is also retained in other ochrophytes (e.g., Ectocarpus) [START_REF] Dorrell | Secondary plastids of stramenopiles[END_REF][START_REF] Corguillé | Plastid genomes of two brown algae, Ectocarpus siliculosus and Fucus vesiculosus: further insights on the evolution of red-algal derived plastids[END_REF], suggesting it was present in the diatom common ancestor and lost from other species. Similarly, the ilvB and ilvH genes, encoding the small and large subunits of acetolactate synthase, have been retained in the chloroplast genomes of the diatom genera Leptocylindrus, Coscinodiscus, Cerataulina, Acanthoceros and Eunotia [START_REF] Yu | Evolution of the plastid genomes in diatoms[END_REF]115). Conversely, the light-independent protochlorophyllide oxidoreductase complex, encoded by the chlB, chlL and chlN genes, is uniquely chloroplast-encoded in the diatom Toxarium (116). These diatoms are distantly positioned to one another and (except for Leptocylindrus) to the base of the diatom tree ( 4), but single-gene phylogenies reveal likely vertical origins of each gene (116), suggesting that they have been independently lost in a wide range of other diatoms.

Fig. 4. Plastid genomes of diatoms and their relatives.

Venn Diagram of chloroplastencoded genes commonly found in photosynthetic diatoms (3), non-photosynthetic diatoms e.g. Nitzschia [START_REF] Kamikawa | Diversity of organellar genomes in non-photosynthetic diatoms[END_REF], ochrophytes assigned to the hypogyristea (pelagophytes, dictyochophytes, Triparma) [START_REF] Han | Dictyochophyceae plastid genomes reveal unusual variability in their organization[END_REF][START_REF] Ong | Analyses of the commplete chloroplast genome sequences of two members of the Pelagophyceae: Aureococcus anophageferrens CCMP1984 and Aureooumbra lagunensis CCMP1507[END_REF][START_REF] Tajima | Sequencing and analysis of the complete organellar genomes of Parmales, a closely related group to Bacillariophyta (diatoms)[END_REF], and chrysista (chrysophytes, Nannochloropsis, phaeophytes) [START_REF] Ševčíková | Updating algal evolutionary relationships through plastid genome sequencing: did alveolate plastids emerge through endosymbiosis of an ochrophyte?[END_REF][START_REF] Cattolico | Chloroplast genome sequencing analysis of Heterosigma akashiwo CCMP452 (West Atlantic) and NIES293 (West Pacific) strains[END_REF][START_REF] Corguillé | Plastid genomes of two brown algae, Ectocarpus siliculosus and Fucus vesiculosus: further insights on the evolution of red-algal derived plastids[END_REF][START_REF] Ševcíková | Plastid genomes and proteins illuminate the evolution of eustigmatophyte algae and their bacterial endosymbionts[END_REF]. Genes identified in fewer than 25% of published genomes within a particular lineage are not shown.

Other diatoms have lost genes typically retained in the chloroplast genomes of other species (Figs. 5,6). Substantial losses of chloroplast-encoded genes have been noted in the centric species Proboscia and the pennate species Astrosyne radiata, which form extremely long branches in diatom chloroplast gene trees [START_REF] Yu | Evolution of the plastid genomes in diatoms[END_REF]117). Astrosyne was isolated from a shallowwater coral reef habitat in Guam (118), whereas Proboscia was isolated from the Red Sea, and it remains to be determined if this reductive evolution is correlated to high-light or temperature adaptations in either species (3). 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 dnaK 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 petF 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 petL 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 petM 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Gene

L e p t o c y l i n d r u s d a n i c u s P r o b o s c i a A c t i n o c y c l u s s u b t i l i s C o s c i n o d i s c u s r a d i a t u s R h i z o s o l e n i a s e t i g e r a G u i n a r d i a s t r i a t a R h i z o s o l e n i a f a l l a x R h i z o s o l e n i a i m b r i c a t a L i t h o d e s m i u m u n d u l a t u m E u n o t o g r a m m a R o u n d i a c a r d i o p h o r a T h a l a s s i o s i r a w e i s s f l o g i i T h a l a s s i o s i r a o c e a n i c a C C M P 1 0 0 5 C y c l o t e l l a s p T h a l a s s i o s i r a p s e u d o n a n a P l a g i o g r a m m o p s i s v a n h e u r c k i i O d o n t e l l a s i n e n s i s T r i c e r a t i u m d u b i u m C e r a t a u l i n a d a e m o n A c a n t h o c e r a s z a c h a r i a s i i C h a e t o c e r o s s i m p l e x T o x a r i u m u n d u l a t u m E C T 3 8 0 2 A t t h e y a l o n g i c o r n i s B i d d u l p h i a b i d d u l p h i a n a B i d d u l p h i a t r i d e n s A s t e r i o n e l l o p s i s g l a c i a l i s P l a g i o g r a m m a s t a u r o p h o r u m A s t e r i o n e l l a f o r m o s a A s t r o s y n e r a d i a t a S y n e d r a a c u s L i c m o p h o r a E u n o t i a n a e g e l i i C y l i n d r o t h e c a c l o s t e r i u m N i t z s c h i a p a l e a H a s l e a n u s a n t a r a S e m i n a v i s r o b u s t a D 6 E n t o m o n e i s F i s t u l i f e r a s o l a r i s P h a e o d a c t y l u m t r i c o r n u t u m D i d y m o s p h e n i a g e m i n a t a H a l a m p h o r a a m e r i c a n a G o m p h o n e i s m i n u t a v a r . c a s s i e a e H a l a m p h o r a c a l i d i l a c u n a H a l a m p h o r a c o f f e a e f o r m i s atpD

psaC 1 0 1 1 1 1 1 1 1 1 1 1 &11 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 psaE 1 0 1 1 1 1 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 psaI 1 1 1 1 1 1 0 0 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 psaM 1 1 1 1 1 1 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 psb28 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 psbI 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 psbZ 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 rbcR 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 rpl34 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 0 1 rpl36 1 0 1 1 1 1 0 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 rps10 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 rps6 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 rps8 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 sufC 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 thiG 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 thiS 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 ycf33 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ycf35 0 0 1 1 1 1 1 0 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 ycf39 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ycf4 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ycf41 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ycf45 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ycf46 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 ycf66 1 1 1 1 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ycf88 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1
acpP 1 1 1 1 1 0 0 1 1 0 0 0 0 0 1 1 1 1 1 1 1 0 1 0 0 1 0 1 0 0 0 1 0 0 0 0 1 0 1 0 0 0 0 0 syfB 1 0 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 0 1 1 0 0 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 ycf42 0 0 1 1 0 1 1 1 0 1 1 1 1 1 1 1 1 1 0 0 0 0 0 1 1 0 1 0 0 1 1 0 0 0 1 1 1 0 1 0 1 0 1 1 ilvB 1 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 ilvH 1 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 petJ 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 chlB 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 chlL 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 chlN 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 serC 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 0 1 0 0 0 1 1 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 tsf 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1 1 1 0 1 0 0 Fig.

Diatom leucoplast genomes

An even more dramatic degree of reduction is known in some diatoms within the genus Nitzschia, which have secondarily lost the capacity for photosynthesis, and may have originated through one (119) or independent evolutionary events [START_REF] Kamikawa | Multiple losses of photosynthesis in Nitzschia (Bacillariophyceae)[END_REF]. These species retain vestigial, non-photosynthetic plastids known as « leucoplasts », which perform essential biosynthetic functions and still retain genomes, but lack plastid-encoded genes associated with photosynthetic functions, e.g., genes for photosystem I, II, cytochrome b 6 f, the Calvin Cycle, and chlorophyll biosynthesis (Fig. 4) [START_REF] Kamikawa | Diversity of organellar genomes in non-photosynthetic diatoms[END_REF]120). Notably, however, the Nitzschia leucoplast genome does retain genes encoding subunits of plastid ATP synthase (Fig. 4), which has been proposed to function in this organelle in the catabolic consumption of ATP, allowing the maintenance of a thylakoid proton gradient that would permit function of the thylakoid Tat protein import complex (120, 121). Comparative analysis of different nonphotosynthetic Nitzschia species reveals largely conserved leucoplast genome content, consistent either with convergent trajectories following independent losses of photosynthesis, or limited divergence in genome content following a single loss of photosynthesis [START_REF] Kamikawa | Diversity of organellar genomes in non-photosynthetic diatoms[END_REF].

The Nitzschia leucoplast genome is notably less reduced in coding content compared to that of other secondarily non-photosynthetic plastids of similar evolutionary origin, e.g., those identified in non-photosynthetic chrysophytes or in apicomplexans [START_REF] Dorrell | Principles of plastid reductive evolution illuminated by nonphotosynthetic chrysophytes[END_REF]122), which have lost a wider range of leucoplast-encoded functions, including ATP synthase. It has been proposed that the osmotrophic feeding strategies of non-photosynthetic Nitzschia spp., may limit their ability to supplement plastidial functions with heterotrophically acquired metabolites, explaining the greater relative functional autonomy of their leucoplasts compared to phagotrophic (chrysophyte) or parasitic (apicomplexan) lineages [START_REF] Dorrell | Principles of plastid reductive evolution illuminated by nonphotosynthetic chrysophytes[END_REF]120). Ultimately, further functional characterisation is required to understand the metabolic contributions of the Nitzschia leucoplast in the context of other, secondarily non-photosynthetic algal species.; which will be aided by the recent completion of a nuclear transcriptome (120) and draft genome [START_REF] Pendergrass | The genome of the nonphotosynthetic diatom, Nitzschia sp.: Insights into the metabolic shift to heterotrophy and the rarity of loss of photosynthesis in diatoms[END_REF].

Why are certain genes lost from diatom chloroplasts?

It remains to be determined what physiological processes underpin the reductive evolution of diatom organelle genomes. This is the case both for genes that have been lost from diatom chloroplasts compared to other algal groups, and genes that have been lost from individual diatom lineages. In other groups of eukaryotes, e.g., plants and dinoflagellates, the loss of genes from the chloroplast genome is correlated to mutation rate and a loss in constraining selective pressure (123, 124), and it is notable that Astrosyne and Proboscia are two of the fastest-evolving diatom species, considering chloroplast-encoded substitution rates (117).

As a test of this principle, we have compared the degree of conservation between proteins encoded with universal, sporadic and occasional presence in diatom chloroplast genomes (Fig. 6). As controls, we have considered the conservation observed between orthologues from non-diatom stramenopiles, and between diatom and non-diatom species (Fig. 6). We note that the proteins with sporadic distributions in diatom species are much more divergent than would be expected compared to both proteins with conserved distributions, and orthologues from non-diatom stramenopiles (Fig. 6). Thus, the genes that are most frequently lost from diatom chloroplast genomes are likely to be those already under relaxed selection in the diatom chloroplast.

Loss versus transfer of diatom organelle genes

Another related question is under what circumstances genes are lost completely from diatom organelles, versus being relocated to the nucleus. Typically, genes are lost from chloroplast and mitochondrial genomes if they no longer perform necessary functions in these organisms.

In contrast, genes may be relocated to the nucleus to permit differential regulation, to protect from elevated chloroplast mutation rates, or to change the stoichiometry of their expression relative to chloroplast-encoded copies (124-126). In the context of diatoms, the open ocean species Thalassiosira oceanica, which is adapted to tolerate chronic iron limitation (127), has been proposed to have relocated the gene petF, encoding the iron-sulfur protein ferredoxin, to the nucleus, to allow its regulation in response to environmental iron availability (128). In another case, the psb28 gene of the centric diatom T. pseudonana has been shown to be present as both chloroplast-and nucleus-encoded copies, suggesting it is midway through a functional chloroplast to nuclear transfer event (129). Psb28 has a non-essential role in photosynthetic complex assembly, but its absence from the cyanobacteria Synechococcus and showing the mean pairwise identities observed by BLAST searches between complete (>90% coverage) protein coding sequences with universal presence (green), identified to be infrequently lost (red), or to have sporadic distributions (serC, tsf, ilvB, ilvH) in diatom chloroplast genomes, plotted against similar pairwise identity scores for pairs of nondiatom stramenopile sequences (left), and pairs of diatom and non-diatom sequences (right). The regression gradients for the chloroplast-encoded proteins with incomplete presences in diatom chloroplasts are much steeper than those with universal presence, i.e., a small decrease in non-diatom chloroplast sequence identity correlates to a much larger decrease in diatom chloroplast sequence identity. This indicates that the chloroplastencoded proteins that are variably present in diatom chloroplasts are more rapidly diverging, even within their chloroplast milieu, than genes that are never lost.

Synechocystis leads to retarded growth under high light conditions (130). It remains to be determined whether the nucleus-encoded copies of these proteins permit photo-adaptation in T. pseudonana.

Gains and transfers of novel diatom organelle ORFs

Finally, alongside the loss and reduction of organelle-encoded genes, diatom chloroplasts and mitochondria possess novel ORFs not identified in other lineages. For example, some pennate diatom chloroplasts are known to contain serC and tsf genes, encoding serine and tyrsoine recombinases (111). These genes are frequently located on plasmids, and may represent the traces of mobile genetic elements integrated into diatom chloroplasts (131, 132).

Other ORFs are either conserved across all diatoms, or indeed all stramenopiles, hence might form ancestral components of stramenopile organelle genomes. Among these is ycf66, a gene found in plant and green algal chloroplast genomes, and conserved in cyanobacteria (133), although it is frequently lost in some lineages (134, 135), and is not known in the chloroplasts of red algae. A phylogeny of ycf66 positions the ochrophytes as a monophyletic group, within the green lineage, as a sister-group to the streptophyte lineage (i.e., land plants and their close relatives), except for the early-branching species Klebsormidium (Fig. 7).

The most parsimonious explanation for this topology is a transfer of the ycf66 gene from the green chloroplast lineage into a common ancestor of the ochrophyte chloroplast, concomitant with its endosymbiotic uptake, which would represent the first known example of a horizontal gene transfer event between two distantly related chloroplast genomes. Understanding the functional consequences of this transfer will depend on characterisation of the physiological role performed by ycf66, which remains unknown. Fig. 7. Evolution of diatom chloroplast ycf66. Consensus MrBayes and RAxML tree, realised under three substitution matrices (GTR, Jones/ JTT, WAG), for a 31 taxa x 93 aa alignment of ycf66 from cyanobacteria and chloroplast genomes. The MrBayes topology supports placement of the ochrophyte sequences within the green algae, as a sister-group to all streptophyte species except Klebsormidium sp.

Concluding remarks

In this chapter, we have explored the phylogenetic context of the diatoms within their broader constituent lineage, the ochrophytes, within the stramenopiles (Fig. 1). Diatoms are evolutionary mosaics, containing complex chloroplasts with complicated evolutionary histories (Fig. 2); and nuclear genomes that have been enriched via by the horizontal and endosymbiotic acquisition of genes with chloroplast and non-chloroplast functions (Table 1). We show that the diatom nucleus is an evolutionary mosaic, supported by genes of prokaryotic, red, green, and other eukaryotic algal origin (Table 1). While the exact numbers, and even the individual evolutionary events that have given rise to these genes remain uncertain, their presence is indelible, and may have contributed unique biological functions to diatom cells.

Alongside this, we have compared reductive trajectories in mitochondrial (Fig. 3) and chloroplast genome content (Figs. 4,5) across the stramenopile tree of life, focusing on sources of evolutionary difference within the diatom lineage. Diatom organelles are marked by reductive evolution, although greater extremes exist in both non-diatom and diatom branches of the stramenopile tree (e.g., non-photosynthetic members of the genus Nitzschia) than those observed in the diatom lineage as a whole. We show that the genes are frequently lost from diatom chloroplast genomes tend to exhibit lower global similarity than wellconserved genes (Fig. 6). It remains to be determined whether this is driven by chloroplast mutation rate, which appears to be somewhat lower in diatoms than that of the mitochondria, despite having a more conserved coding content (136) Figs. [START_REF] Parks | Signal, uncertainty, and conflict in phylogenomic data for a diverse lineage of microbial eukaryotes (diatoms, Bacillariophyta)[END_REF][START_REF] Ichinomiya | Diversity and oceanic distribution of the Parmales (Bolidophyceae), a picoplanktonic group closely related to diatoms[END_REF]; or via relaxed selection pressure, which has been shown to substantially vary across different chloroplast genes in other algal lineages (e.g., dinoflagellates (137)).

Finally, a possible gain of a chloroplast-encoded function has been identified, the uncharacterised open reading frame ycf66, which appears to have been acquired by ochrophyte chloroplasts via horizontal gene transfer of an equivalent open reading frame from green algal chloroplasts (Fig. 7). The role of this transfer event awaits functional and environmental characterisation. We stress the importance of considering function alongside the number, tempo, and mode of acquisition of novel diatom genes, when exploring the extraordinary environmental success of this lineage. In this context, emergent transformable systems (138-140), and environmental sequence datasets (141, 142), may cast new functional insights into the consequences of dynamic evolutionary events in diatom nuclei and organellular genomes.

Fig. 2 . 14 Fig. 3 .Fig. 4 . 16 Fig. 5 .

 21434165 Fig. 2. Schematic diagram of the diatom chloroplast ...………………………………...….… 7Table 1. Sizes and sources of gene transfers into and out of diatom nuclear genomes ........ 8-9 Methods and problems when exploring origins of diatom nuclear genes ……..……………... Prokaryotic signals .……………………………………………………………………..…... Red algal-related signals ..…………………………………………………………………... 12 Green algal-derived signals ……………………………………………………………..….. Have diatoms donated genes to other organisms? .….…………………………………..…. 14

Fig. 7 .

 7 Fig. 7. CCTH. (a) Telonemid: Telonema subtile; (b) Haptophyte: Pavlova pinguis; (c) Haptophyte, coccolithophorid: Emiliania huxleyi; (d) Cryptophyte; Cryptomonas sp.; (e) Kathablepharid: Kathablepharis sp.; (f) Centrohelid: Heterophrys sp. Scale bar in f: for a, 5 μm; b, 20 μm; c-f, 10 μm.

Fig. 7 .

 7 Fig. 7. CCTH. (a) Telonemid: Telonema subtile; (b) Haptophyte: Pavlova pinguis; (c) Haptophyte, coccolithophorid: Emiliania huxleyi; (d) Cryptophyte; Cryptomonas sp.; (e) Kathablepharid: Kathablepharis sp.; (f) Centrohelid: Heterophrys sp. Scale bar in f: for a, 5 μm; b, 20 μm; c-f, 10 μm.

Fig. 2 .

 2 Fig. 2. Schematic diagram of the diatom chloroplast, adapted from (1, 2) ; demonstrating the ulstrastructure of the chloroplast, and the major evolutionary classes of chloroplast-and nucleus-encoded chloroplast proteins.

Fig. 3 .

 3 Fig. 3. Organisation of the diatom mitogenome. Top: Venn Diagram of mitochondrial genome-encoded proteins assignable to the common ancestors of diatoms, other ochrophytes (chrysista), and aplastidic stramenopiles (oomycetes, labyrinthulomycetes, biocosiceds). Bottom : heatmap of the occurrence of seven genes with sporadic distribution in published diatom mitochondrial genomes. Red cells indicate presence and blank cells indicate absence.

Fig. 6 .

 6 Fig.6. Dynamic of evolutionary rates in diatom chloroplast genomes. Scatterplots showing the mean pairwise identities observed by BLAST searches between complete (>90% coverage) protein coding sequences with universal presence (green), identified to be infrequently lost (red), or to have sporadic distributions (serC, tsf, ilvB, ilvH) in diatom chloroplast genomes, plotted against similar pairwise identity scores for pairs of nondiatom stramenopile sequences (left), and pairs of diatom and non-diatom sequences (right). The regression gradients for the chloroplast-encoded proteins with incomplete presences in diatom chloroplasts are much steeper than those with universal presence, i.e., a small decrease in non-diatom chloroplast sequence identity correlates to a much larger decrease in diatom chloroplast sequence identity. This indicates that the chloroplastencoded proteins that are variably present in diatom chloroplasts are more rapidly diverging, even within their chloroplast milieu, than genes that are never lost.

Table 1 . Sizes and sources of gene transfers into and out of diatom nuclear genomes

 1

	Source	Method	Bacterial genes	Red genes	Green genes	Other genes
	Armbrust et	First annotation of	n.a.	182 genes with	865 genes	806 genes with
	al. (2004)	T. pseudonana		exclusive	with exclusive	exclusive
	(54)	(Tp) genome ;		homology to	homology to	homology to Mus
		BLAST		Cyanidioschyzon	Arabidopsis	musculus
		comparison to		merolae (red algal	thaliana
		plant, red algae		reference)
		and animal
		genomes
	Bowler et	First annotation of	784, of which	171, of which 160	n.a.	n.a.
	al. (2008)	P. tricornutum	587 unique to	only present in
	(55)	(Pt) genome ;	Pt; 267 shared	ochrophytes
		automated	with Tp ; and 73
		phylogenies	conserved
			across
			stramenopiles
	Moustafa et	Automated	n.a.	418 in Pt ; 460 in	1,757 in Pt ;	248 Pt and 211
	al. (2009)	phylogenies of Pt		Tp	1,862 in Tp;	Tp genes of
	(56)	and Tp			647 and 716	ambiguous green/
		genomes; C.			respectively	red origin
		merolae only			of
		complete red			prasinophyte
		model			origin
	Deschamps	Manual	n.a.	n.a.	89 « green »	n.a.
	and Moreira	reannotation of			trees in (56)
	(2012) (66)	data from (56)			consistent
					with green
					origins ; 91
					with red
					origins;
					remainder
					unresolved
	Dorrell et al.	Manual	26/ 502 (5%)	255/502 (50%)	105/ 502	243 inferred
	(2017) (39)	phylogenies of	tractable	tractable	(20%)	transfers from
		770 plastid	proteins	proteins ; of	tractable	ochrophytes to
		proteins		which only 4	proteins ;	haptophytes ;
		conserved across		shared with	majority	most likely from
		stramenopiles		oomycetes	(76%) suggest	pelagophyte/
		with densely			position in	dictyochophyte
		sampled (genome			chlorophytes	ancestor
		and			to exclusion
		transcriptome)			of
		reference dataset			streptophytes
	Rastogi et	BLAST best hit	584, of which	459, of which 50	1,981, of	882 genes of
	al. (2018)	annotation of	133 tractable to	tractable to	which 181	inferred
	(65)	version 3 Pt	diatom common	diatom common	tractable to	amorphean
		genome with	ancestor, 226 to	ancestor, 353 to	diatom	(animal, fungi,

Acknowledgments

RGD acknowledges a CNRS Momentum Fellowship (awarded 2019-2021).