Anna M G Novák Vanclová

Charlotte Nef

Adél Vancl
email: vanclova@gmail.com

Fuhai Liu

Zoltán Füssy

Chris Bowler

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

Divergent and diversified proteome content across a serially acquired plastid lineage

Keywords: Myzozoans, Protist, Transcriptomics, Automated Tree Sorting, Post-endosymbiotic organelle evolution, Shopping Bag Model

Dinoflagellates are a diverse group of ecologically significant micro-eukaryotes, prone to the loss and replacement of their plastids via serial endosymbiosis. One such replacement took place in the ancestors of the Kareniaceae, which harbor haptophyte-derived plastids containing the pigment fucoxanthin, instead of the ancestral peridinin dinoflagellate plastid. The metabolic functions of the fucoxanthin plastid are performed by a diverse range of nucleus-encoded and plastid-targeted proteins, which may originate from the haptophyte ancestor of the fucoxanthin plastid, the peridinin-containing ancestor of the dinoflagellate host, and/or from lateral gene transfers. However, the evolutionary composition of fucoxanthin plastid proteomes across the diversity of kareniacean dinoflagellates remains poorly understood. Here, we determine the total composition of the plastid proteomes of seven distantly-related kareniacean dinoflagellates, including newly-sequenced members of three genera (Karenia, Karlodinium, and Takayama). Using a custom plastid-targeting predictor, automatic single-gene tree building and phylogenetic sorting of plastid-targeted proteins, we project relatively limited (~10%) and functionally distinctive contributions of the haptophyte endosymbiont to the fucoxanthin plastid proteome, in comparison to plastid-targeted proteins of dinoflagellate host origin. Considering a concatenated multigene phylogeny of haptophyte-derived plastid proteins, we show that the haptophyte order Chrysochromulinales is the closest living relative of the fucoxanthin plastid donor. We additionally perform detailed analyses of the N-terminal targeting sequences of kareniacean plastid signal peptides, reporting a surprisingly high sequence conservation. Finally, considering planetary-scale distributions of key kareniacean genera and haptophyte orders from Tara Oceans, we suggest ecological and mechanistic factors accompanying the endosymbiotic acquisition of the fucoxanthin plastid.

Introduction

The origin of oxygenic photosynthesis, and its adoption by eukaryotes, has fundamentally changed the evolutionary landscape of life. Originating in cyanobacteria, photosynthesis has spread across the eukaryotes through multiple endosymbiotic acquisitions of plastids (or chloroplasts). These began with the primary plastids of cyanobacterial origin that represent the defining synapomorphy of Archaeplastida (glaucophytes, red and green algae, and plants) and are surrounded by two membranes. Plastids subsequently spread into other groups (e.g., diatoms, haptophytes, dinoflagellates, euglenids) through multiple endosymbiotic acquisitions of eukaryotic algae, forming "complex plastids" that possess additional membranes, often contained within the endoplasmic reticulum. Across the tree of life, plastids provide their hosts with novel metabolic and biosynthetic capabilities and serve as important vectors of horizontal gene transfer between different algal lineages [START_REF] Howe | The origin of plastids[END_REF][START_REF] Ku | Endosymbiotic origin and differential loss of eukaryotic genes[END_REF][START_REF] Vanclová | Metabolic quirks and the colourful history of the Euglena gracilis secondary plastid[END_REF][START_REF] Dorrell | Phylogenomic fingerprinting of tempo and functions of horizontal gene transfer within ochrophytes[END_REF].

Plastids have their own genomes that typically encode core photosynthetic proteins alongside some of their genetic housekeeping machinery and primary metabolic enzymes. Their size and coding capacity vary, typically possessing 20-250 genes, driven by gradual and somewhat convergent gene loss [START_REF] Uthanumallian | Tightly Constrained Genome Reduction and Relaxation of Purifying Selection during Secondary Plastid Endosymbiosis[END_REF]. Plastids however require a much larger inventory of proteins to function (e.g., > 2000 in Arabidopsis and model eukaryotic algal groups; [START_REF] Kleffmann | The Arabidopsis thaliana Chloroplast Proteome Reveals Pathway Abundance and Novel Protein Functions[END_REF][START_REF] Gruber | Plastid proteome prediction for diatoms and other algae with secondary plastids of the red lineage[END_REF][START_REF] Vanclová | Metabolic quirks and the colourful history of the Euglena gracilis secondary plastid[END_REF]; and most of the individual plastidial proteins are encoded in the nucleus and post-translationally imported based on a cleavable N-terminal targeting signal. These plastidtargeted proteins comprise the expression products of plastid-derived genes that were transferred to host nucleus, host-derived genes newly adapted for plastidial function, and genes originating from lateral gene transfer (LGT) from other sources, including other symbionts that the host has interacted with over its evolutionary history [START_REF] Moustafa | Chlamydiae Has Contributed at Least 55 Genes to Plantae with Predominantly Plastid Functions[END_REF]. This makes all plastid proteomes innately chimeric entities, or "shopping bags", whose composition may reflect both general principles of organelle evolution and the unique evolutionary histories of individual lineages [START_REF] Larkum | Shopping for plastids[END_REF].

Dinoflagellates are a diverse group of unicellular algae that are ecologically and economically significant; including crucial symbionts of corals, and toxin-producing species that can cause major losses in fisheries and endanger public health [START_REF] Hackett | Dinoflagellates: a remarkable evolutionary experiment[END_REF][START_REF] Wang | Neurotoxins from Marine Dinoflagellates: A Brief Review[END_REF]. Within the eukaryotic tree of life, they fall among the supergroup Alveolata, specifically Myzozoa along with chrompodellids and apicomplexans (Ševčíková et al., 2015). Dinoflagellates are of further evolutionary interest given their unique and idiosyncratic cell biology. Their nuclear genomes are generally extremely large (up to several hundred Gbp) with permanently condensed chromosomes that lack histones, while their mitochondrial genomes are very streamlined [START_REF] Lin | Genomic understanding of dinoflagellates[END_REF][START_REF] Dorrell | Integration of plastids with their hosts: Lessons learned from dinoflagellates[END_REF]. The evolutionarily ancestral dinoflagellate plastids are descended from red algae and bound by three membranes [START_REF] Moog | Cellular compartmentation follows rules: The Schnepf theorem, its consequences and exceptions[END_REF]. The ancestral dinoflagellate plastid is further marked by multiple functional oddities, including a unique carotenoid pigment, peridinin [START_REF] Haxo | Peridinin-Chlorophyll a Proteins of the Dinoflagellate Amphidinium carterae[END_REF]; and a bacterial-like nucleus-encoded form II RuBisCO [START_REF] Morse | A nuclear-encoded form II RuBisCO in dinoflagellates[END_REF] in contrast to the partially plastid-encoded form I RuBisCO found in all other plastid lineages. Dinoflagellate plastid genomes are highly fragmented and organized into small unigenic elements termed "minicircles". These transcripts undergo distinctive maturation events, including extensive substitutional editing and the addition of a 3' poly(U) tail not found in most other plastid lineages [START_REF] Zauner | Substitutional editing of transcripts from genes of cyanobacterial origin in the dinoflagellate Ceratium horridum[END_REF]Wang and Morse, 2006;[START_REF] Dorrell | Integration of plastids with their hosts: Lessons learned from dinoflagellates[END_REF].

Dinoflagellates as a group are ecologically opportunistic: most plastid-bearing representatives are in fact mixotrophic [START_REF] Cohen | Dinoflagellates alter their carbon and nutrient metabolic strategies across environmental gradients in the central Pacific Ocean[END_REF][START_REF] Jeong | Feeding diverse prey as an excellent strategy of mixotrophic dinoflagellates for global dominance[END_REF] and in global meta-genomic analyses (e.g., Tara Oceans) dinoflagellates appear mostly as predators and parasites [START_REF] Pierella Karlusich | Biogeographical Patterns and Genomes of Aquatic Photoautotrophs[END_REF]. Their plastids are frequently secondarily reduced to non-photosynthetic organelles, lost completely [START_REF] Saldarriaga | Dinoflagellate Nuclear SSU rRNA Phylogeny Suggests Multiple Plastid Losses and Replacements[END_REF][START_REF] Sanchez-Puerta | Plastid Genes in a Non-Photosynthetic Dinoflagellate[END_REF][START_REF] Gornik | Endosymbiosis undone by stepwise elimination of the plastid in a parasitic dinoflagellate[END_REF][START_REF] Cooney | Phylogenomics shows unique traits in Noctilucales are derived rather than ancestral[END_REF], or replaced in new symbiogenetic events. These may result in fully integrated plastids [START_REF] Tengs | Phylogenetic analyses indicate flint the 19'hexanoyloxy-fucoxanthincontaining dinoflagellates have tertiary plastids of haptophyte origin[END_REF][START_REF] Matsumoto | Greencolored Plastids in the Dinoflagellate Genus Lepidodinium are of Core Chlorophyte Origin[END_REF], complex endosymbionts retaining plastids, mitochondria, and nuclei [START_REF] Imanian | The dinoflagellates Durinskia baltica and Kryptoperidinium foliaceum retain functionally overlapping mitochondria from two evolutionarily distinct lineages[END_REF][START_REF] Sarai | Dinoflagellates with relic endosymbiont nuclei as models for elucidating organellogenesis[END_REF], and kleptoplastids of various degrees of stability [START_REF] Koike | A novel type of kleptoplastidy in Dinophysis (Dinophyceae): Presence of haptophyte-type plastid in Dinophysis mitra[END_REF][START_REF] Myung | First successful culture of the marine dinoflagellate Dinophysis acuminata[END_REF][START_REF] Gast | Kleptoplasty in an Antarctic dinoflagellate: Caught in evolutionary transition?[END_REF]. In some cases, the co-existence of a new organelle or endosymbiont with a remnant of the ancestral plastid has been proposed [START_REF] Hehenberger | A kleptoplastidic dinoflagellate and the tipping point between transient and fully integrated plastid endosymbiosis[END_REF], and in one lineage, a complete repurposing of parts of the plastid and mitochondrion into an enigmatic, photosynthesisunrelated optical structure termed "ocelloid" took place [START_REF] Gavelis | Eyelike ocelloids are built from different endosymbiotically acquired components[END_REF].

Kareniaceae, typified by the genera Karenia, Karlodinium, and Takayama, are mixotrophic dinoflagellates whose plastids are related to those of haptophytes. Kareniaceae are the most abundant dinoflagellate lineage with serially acquired plastids in Tara Oceans (Table S1, based on data from [START_REF] Engelen | Eukaryotic plankton diversity in the sunlit ocean[END_REF], and both Karenia and Karlodinium have important impacts as toxic components of harmful algal blooms. Further ecological and evolutionary complexity found within the Kareniaceae is the Antarctic native Ross Sea dinoflagellate (RSD) [START_REF] Gast | Kleptoplasty in an Antarctic dinoflagellate: Caught in evolutionary transition?[END_REF][START_REF] Hehenberger | A kleptoplastidic dinoflagellate and the tipping point between transient and fully integrated plastid endosymbiosis[END_REF], which uses a kleptoplastid from the haptophyte Phaeocystis antarctica acquired independently to that of other Kareniaceae.

Instead of peridinin, plastids of the Kareniaceae contain the accessory pigment fucoxanthin typical of their haptophyte ancestors [START_REF] Zapata | Photosynthetic pigments in 37 species (65 strains) of Haptophyta: implications for oceanography and chemotaxonomy[END_REF][START_REF] Zapata | Pigment-based chloroplast types in dinoflagellates[END_REF], and are bound by four membranes, the outermost of which is continuous with the endoplasmic reticulum as per haptophytes, and distinct from the three membrane-bound plastids found in peridinin dinoflagellates.

Partial kareniacean plastid genomes and transcriptomes have been sequenced for the species Karlodinium micrum (syn. veneficum) and Karenia mikimotoi, indicating the retention of around 100 genes on a single circular or linear chromosome [START_REF] Gabrielsen | Genome Evolution of a Tertiary Dinoflagellate Plastid[END_REF][START_REF] Dorrell | Diversity of transcripts and transcript processing forms in plastids of the dinoflagellate alga Karenia mikimotoi[END_REF], which is somewhat fewer than the ca. 140 genes associated with haptophyte plastids, alongside the presence of episomal minicircles [START_REF] Richardson | Genome-Wide Transcript Profiling Reveals the Coevolution of Plastid Gene Sequences and Transcript Processing Pathways in the Fucoxanthin Dinoflagellate Karlodinium veneficum[END_REF]. Further studies, based on expressed sequence tags (ESTs) [START_REF] Ishida | Second-and third-hand chloroplasts in dinoflagellates: Phylogeny of oxygen-evolving enhancer 1 (PsbO) protein reveals replacement of a nuclear-encoded plastid gene by that of a haptophyte tertiary endosymbiont[END_REF][START_REF] Nosenko | Chimeric plastid proteome in the Florida "red tide" dinoflagellate Karenia brevis[END_REF][START_REF] Patron | A tertiary plastid uses genes from two endosymbionts[END_REF] and, more recently, transcriptomes of Karenia brevis and Karlodinium micrum realized through the Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP) [START_REF] Burki | Endosymbiotic gene transfer in tertiary plastid-containing dinoflagellates[END_REF][START_REF] Keeling | The Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP): Illuminating the Functional Diversity of Eukaryotic Life in the Oceans through Transcriptome Sequencing[END_REF][START_REF] Matsuo | Patterns in evolutionary origins of heme, chlorophyll a and isopentenyl diphosphate biosynthetic pathways suggest non-photosynthetic periods prior to plastid replacements in dinoflagellates[END_REF][START_REF] Hehenberger | A kleptoplastidic dinoflagellate and the tipping point between transient and fully integrated plastid endosymbiosis[END_REF], have provided foundational insights into the nucleus-encoded proteome of the kareniacean plastid. These are defined by a bipartite targeting sequence, consisting of an N-terminal signal peptide followed by a hydrophilic transit peptide, similar to those of haptophytes, although with apparent differences in composition [START_REF] Patron | Transit peptide diversity and divergence: A global analysis of plastid targeting signals[END_REF][START_REF] Yokoyama | Evolutionary analyses of the nuclear-encoded photosynthetic gene psbo from tertiary plastid-containing algae in dinophyta[END_REF]. Phylogenetic analysis of the genes encoding plastid-targeted kareniacean proteins [START_REF] Nosenko | Chimeric plastid proteome in the Florida "red tide" dinoflagellate Karenia brevis[END_REF][START_REF] Patron | A tertiary plastid uses genes from two endosymbionts[END_REF][START_REF] Burki | Endosymbiotic gene transfer in tertiary plastid-containing dinoflagellates[END_REF][START_REF] Bentlage | Complex Ancestries of Isoprenoid Synthesis in Dinoflagellates[END_REF][START_REF] Matsuo | Patterns in evolutionary origins of heme, chlorophyll a and isopentenyl diphosphate biosynthetic pathways suggest non-photosynthetic periods prior to plastid replacements in dinoflagellates[END_REF] indicate that some resolve with haptophytes (i.e., the endosymbiont ancestor), while others show alternative origins. These include key elements of the plastid gene expression machinery (e.g., RNA editing and poly(U) tail addition), which are likely to have been derived from the dinoflagellate host and potentially associated with the ancestral peridinin plastid [START_REF] Dorrell | Functional remodeling of RNA processing in replacement chloroplasts by pathways retained from their predecessors[END_REF][START_REF] Jackson | A tertiary plastid gains RNA editing in its new host[END_REF]; and cofactor (e.g., isopentenyl pyrophosphate and protoporphyrin/haem) biosynthetic pathways, which resolve principally either with dinoflagellates or sources suggesting horizontal acquisition [START_REF] Matsuo | Patterns in evolutionary origins of heme, chlorophyll a and isopentenyl diphosphate biosynthetic pathways suggest non-photosynthetic periods prior to plastid replacements in dinoflagellates[END_REF].

To date, a reconstruction of the entire fucoxanthin plastid has yet to be attempted, and its overall metabolic composition and specific evolutionary origin within the haptophytes remain unresolved [START_REF] Yoon | A single origin of the peridinin-and fucoxanthin-containing plastids in dinoflagellates through tertiary endosymbiosis[END_REF][START_REF] Takahashi | Gertia stigmatica gen. et sp. nov. (Kareniaceae, Dinophyceae), a New Marine Unarmored Dinoflagellate Possessing the Peridinin-type Chloroplast with an Eyespot[END_REF][START_REF] Leblond | Galactolipid composition of Gertia stigmatica ( Dinophyceae ), an atypical member of the Kareniaceae with a peridinin-containing , nonhaptophyte-derived plastid[END_REF]. Here, we present a holistic reconstruction of the pan-kareniacean plastid proteome across seven model species, integrating newly sequenced transcriptomic datasets for two additional strains of Karenia and Karlodinium and the first transcriptome for the genus Takayama, in silico identification and phylogenomic analysis of genes encoding plastid-targeted proteins.

These data elucidate the metabolic functions of the fucoxanthin plastid in its kareniacean host, its deep phylogenetic origins, and the evolutionary principles that may have constrained its genesis and early history. In particular, we emphasize the significance of integration with pre-existing components and systems inherited from the host and its previous plastid. Our study illuminates the deep evolutionary history of this ecologically important algal lineage, and some of the functional principles that may underpin symbiogenetic plastid establishment across eukaryotes.

Results

Pan-transcriptomics reveals the core fucoxanthin plastid proteome

Five new transcriptomic datasets were produced for fucoxanthin dinoflagellates: Karenia mikimotoi and Karenia papilionacea, the RCC3446 strain of Karlodinium micrum and its distant relative Karlodinium armiger, and for Takayama helix, a predator of other dinoflagellates more closely related to Karlodinium than Karenia [START_REF] Jeong | Mixotrophy in the phototrophic dinoflagellate Takayama helix (family Kareniaceae): Predator of diverse toxic and harmful dinoflagellates[END_REF], for which to our knowledge this is the first reported transcriptomic dataset. These libraries, alongside four previously sequenced MMETSP libraries for Karenia brevis, one for Karlodinium micrum strain CCMP2283, and an independently sequenced transcriptome for RSD, represent the pan-kareniacean library used for all downstream analyses.

The raw dataset sizes range from around 200 to 660 Mbp but contain large amounts of redundant or partially redundant transcripts, reflecting the extensive paralogization and pseudogenization associated with dinoflagellate nuclear genomes [START_REF] Stern | Environmental Barcoding Reveals Massive Dinoflagellate Diversity in Marine Environments[END_REF]. To reduce the size and redundancy of our datasets for subsequent analysis, we selected the most complete paralogs of each studied protein by cd-hit. The completeness of the datasets as assessed by BUSCO is around 70% which is rather high in comparison to other dinoflagellate datasets (Table 1, Table S2).

Guided in silico prediction reveals within-lineage divergence in fucoxanthin plastid proteome contents

A modified version of ASAFind [START_REF] Gruber | Plastid proteome prediction for diatoms and other algae with secondary plastids of the red lineage[END_REF], which has previously been used to reconstruct in silico the haptophyte plastid proteome [START_REF] Dorrell | Chimeric origins of ochrophytes and haptophytes revealed through an ancient plastid proteome[END_REF], was constructed with a custom scoring matrix for fucoxanthin dinoflagellate plastids (see Materials and Methods) and used in combination with SignalP 5.0 in the final prediction pipeline. After preliminary automatic annotation of the retrieved in silico plastidial proteomes of all studied organisms by KAAS (https://www.genome.jp/kegg/kaas/; [START_REF] Moriya | KAAS: an automatic genome annotation and pathway reconstruction server[END_REF], we noticed that some of the integral plastidial proteins missing from these datasets could be captured by an alternative prediction approach combining PrediSI [START_REF] Hiller | PrediSi: Prediction of signal peptides and their cleavage positions[END_REF] and ChloroP [START_REF] Emanuelsson | ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites[END_REF] employed previously for Euglena gracilis data [START_REF] Ebenezer | Euglena gracilis Genome and Transcriptome: Organelles, Nuclear Genome Assembly Strategies and Initial Features[END_REF], and so included these as well. The proportion of redundancy-treated translated transcripts predicted as plastidtargeted ranged from 7.5 to 14.5 % in K. micrum and K.brevis, respectively (~9.5 % being both average and modal value). Of these, most have a targeting sequence predictable by modified ASAFind, while between 16 to 22% represent additions based solely on the abovementioned alternative signal.

The putative phylogenetic origin of all predicted plastid proteins was investigated via a custom pipeline, integrating homology mining, single-gene tree construction and sorting based on topology and its statistical support (Figure S1). Between 40 and 55 % of each proteome dataset had some homologs in our reference database containing representatives for most major eukaryotic groups, particularly enriched in haptophyte and dinoflagellate transcriptomes (see Materials and Methods). In about one third of these, the identified homologs were shared solely with other dinoflagellates and were therefore considered dinoflagellate-specific proteins without any further phylogenetic analysis. For the rest, single-gene trees (29,329 in total) were constructed and automatically sorted based on which evolutionary origin they support. The main two investigated categories were "plastid-early" (i.e., vertically inherited from the dinoflagellate host; colour-coded as blue and purple dependent on whether the protein retrieved only dinoflagellate homologues or showed deeper homology to other alveolates or myzozoans lineages) and "plastid-late" (i.e., immediately clustering with the haptophyte endosymbiont; colour-coded as orange in all graphical materials). In addition to this, potential cases of LGT from prokaryotes, green algae and ochrophytes were identified.

Of all the proteins with homologs in the database, 65-80% were categorized as "plastid-early" in all studied organisms (Figure 1A). The proportion of proteins categorized as "plastid-late" is generally in accord with the previously published estimates [START_REF] Burki | Endosymbiotic gene transfer in tertiary plastid-containing dinoflagellates[END_REF] but there was a noticeable difference between the genera, with approximately 15% noted in all three species of Karenia, 10% in Karlodinium, and 6% in Takayama. The proportion of proteins shared and clustering with ciliates, i.e., primarily heterotrophic alveolates ("non-plastid signal"), is almost negligible in all studied organisms and so is the contribution of prokaryotic or "green" LGT (cases of which do not exceed 20 per organism). The amount of "brown" LGT is slightly higher than the amount of non-plastid signal (varying from 12 to 107 per organism). Between 10 and 20 % of signal in each organism was not sortable into any of the investigated categories.

Phylogenomics reveals a chrysochromulinacean and potentially incongruent origin of the fucoxanthin plastid

Previous studies of the fast-evolving fucoxanthin plastid genome revealed a deep-branching position sister to all prymnesiophytes but no affinity towards individual orders [START_REF] Choi | Newly discovered deep-branching marine plastid lineages are numerically rare but globally distributed[END_REF][START_REF] Klinger | Plastid Transcript Editing across Dinoflagellate Lineages Shows Lineage-Specific Application but Conserved Trends[END_REF][START_REF] Kawachi | Rappemonads are haptophyte phytoplankton[END_REF]. To recover a more specific affinity, we enumerated the plastid-targeted proteins clustering specifically with one of six haptophyte families (Chrysochromulinales, Isochrysidales, Phaeocystales, Coccolithales, Pavlovales, and Prymnesiales; Figure 1B), constituting approximately onequarter of the total plastid-late signal.

Across all tree topologies, we note of selected tree topologies revealed frequent apparent non-monophyly of the kareniacean genes, with Takayama sequences often branching separately from the others. Nonetheless, for each of the seven studied fucoxanthin-containing Kareniaceans the most abundant category (up to >50%) was the Chrysochromulinales. A concatenation of 22 genes that were universally identified as plastid-late across all fucoxanthin genera, independent of deeper haptophyte origin, and were either present in a single copy or had only a few easily identifiable and removable paralogs, further robustly placed the fucoxanthin plastid as a sister-group to the Chrysochromulinales (sp. Chrysochromulina brevifilum, Chrysochromulina rotalis and Chrysochromulina sp.; 100% bootstrap support), to the exclusion of the closest related haptophyte taxa within the Prymnesiales (Figure 2A).

The plastid-late proteins from the RSD, which possess a kleptoplastid derived from Phaeocystis antarctica, resolve clearly with Phaeocystis anatarctica within the Phaeocystales, both considering single gene-trees (>70%) and the concatenated topology (Figures 12). We further noted a strong secondary signal to the family Isochrysidales in all kareniacean species including the RSD, even normalizing for dataset size (Figure S2), although many were specific to individual genera (Figure S3). Finally, we searched the new transcriptomes for contigs potentially representing plastid transcripts (recovering 13 to 39 genes in each case) and reconstructed a preliminary plastid phylogeny based concatenated nucleotide sequences. Consistent with previous studies, the fucoxanthin plastid genomes resolved as a long-branch sister group to all Prymnesiophyceae, although with a notable separation of Takayama from Karenia and Karlodinium (Figure S4) [START_REF] Choi | Newly discovered deep-branching marine plastid lineages are numerically rare but globally distributed[END_REF][START_REF] Kawachi | Rappemonads are haptophyte phytoplankton[END_REF].

Finally, we used the whole transcriptomes as input for the PhyloFisher pipeline [START_REF] Tice | PhyloFisher: A phylogenomic package for resolving eukaryotic relationships[END_REF] to construct a matrix of 241 pan-eukaryotic nuclear-encoded genes to obtain a reference phylogeny of the studied kareniacean nuclear genomes in the context of full eukaryotic tree of life. The topology reconstructed from PhyloFisher matrix is consistent with recently published rRNA trees [START_REF] Takahashi | Gertia stigmatica gen. et sp. nov. (Kareniaceae, Dinophyceae), a New Marine Unarmored Dinoflagellate Possessing the Peridinin-type Chloroplast with an Eyespot[END_REF][START_REF] Ok | Shimiella gen. nov. and Shimiella gracilenta sp. nov. (Dinophyceae, Kareniaceae), a Kleptoplastidic Dinoflagellate from Korean Waters and its Survival under Starvation[END_REF] with Karenia as sister to both Karlodinium and Takayama (Figure 2B), we note however that this phylogenetic topology differs to those reconstructed with both the plastid-late and plastid-encoded genes, which place Karenia and Karlodinium together, to the exclusion of Takayama (Figure 2; Figure S4). The monophyletic grouping of Karlodinium and Takayama was robustly rejected by approximately unbiased (AU) tests of the plastid-late and plastid-encoded tree topologies (p=0.006). The apparent incongruence between the placement of the Takayama plastid in the plastid-late, plastid-encoded and concatenated nuclear topologies raises questions concerning its endosymbiotic origin relative to other fucoxanthin-containing lineages.

A hybrid plastid proteome with species-specific innovations

The plastid proteins of each kareniacean were automatically annotated with enzymatic functions based mainly on KEGG annotations, presented as a pivot table (Table S4). Considering χ² enrichments of individual KEGG annotations (KO IDs) across all seven fucoxanthin-containing species, we noted a statistically significant enrichment (p < 0.01) in the plastid-late signal in the BRITE category "Photosynthesis", combined category "Translation", and KEGG Pathway "Porphyrin and chlorophyll metabolism", while the plastid-early signal was enriched in BRITE categories "Membrane trafficking", "Protein kinases, phosphatases, and associated proteins", and "Ion channels". Reflecting the probable plastid-early origin of the fucoxanthin plastid RNA processing machinery, categories relating to transcription and mRNA are not biased in their origin.

When analysed manually and qualitatively, additional evolutionary patterns were apparent in the overall metabolic map (Figure 3), some of which were previously described in K. brevis and K. micrum [START_REF] Matsuo | Patterns in evolutionary origins of heme, chlorophyll a and isopentenyl diphosphate biosynthetic pathways suggest non-photosynthetic periods prior to plastid replacements in dinoflagellates[END_REF]. These include notable differences between the genera and species in some pathways or enzymes, and several general particularities such as unexpectedly missing, duplicated, or laterally gained proteins, with full details supplied in Text S6 -Supplementary results and discussion.

Reflecting KEGG enrichments, photosystems, light-harvesting complexes, photosynthetic electron transport chain and ATP synthase subunits are almost exclusively of plastid-late origin. So are ribosomal proteins and identified components of the protein import pathways, i.e., the "protein-level" housekeeping of the newest organelle, while the transcription machinery (when not directly plastid-coded, like RNA polymerase) is of mixed origin (see above). Furthermore, many of the pathways closely associated with photosynthetic function (i.e., syntheses of photosynthetic or photoprotective pigments) are of plastid-late origin, whereas the backbone pathways necessary for their synthesis (namely the tetrapyrrole pathway, preceding chlorophyll synthesis, and the MEP/DOXP pathway for synthesis of the IPP precursor of carotenoids) are of mixed or mostly plastid-early origin. Notably, we found almost no plastidial pathway to be of purely plastid-early origin, except perhaps the partial synthesis of some amino acids (Ala, Asp, Lys).

We report small numbers of conserved LGTs from other algal groups or bacteria, which are particularly concentrated among amino acyl-tRNA synthetases (HARS, TARS, MARS, NARS, PARS; see Figures S5.1-2 for selected exemplary trees), alongside with specific components of individual biosynthetic pathways (green SQD2, HST, haem oxygenase, and brown acetyl-CoA carboxylase, 15-cis-phytoene synthase, zeaxanthin epoxidase; exemplary trees in Figures S5.3-5). These genes were frequently detected across all fucoxanthin genera with strong bootstrap support and are therefore unlikely to result from contamination in individual transcriptomes or phylogenetic artifact. Some otherwise broadly conserved proteins were not detected in any kareniacean transcriptomes, most notably SQD1, one of the two enzymes for the synthesis of essential plastidial glycerolipid sulfoquinovosyldiacylglycerol (SQDG), and the delta subunit of the photosynthetic ATP synthase (atpH, K02113). The former may be due to a very low expression level as it was previously missed in various algae even in dedicated studies [START_REF] Riccio | Monogalactosyldiacylglycerol and sulfolipid synthesis in microalgae[END_REF]. The latter is more baffling as this conserved and essential subunit is usually plastid-encoded but was not retrieved as such in the previous plastid genome analyses [START_REF] Gabrielsen | Genome Evolution of a Tertiary Dinoflagellate Plastid[END_REF][START_REF] Dorrell | Diversity of transcripts and transcript processing forms in plastids of the dinoflagellate alga Karenia mikimotoi[END_REF] and was not identified in any of the full transcriptomes either. However, an unrelated and structurally dissimilar but functionally analogous subunit of mitochondrial ATPase (ATP5D, K02134) seems to be duplicated and retargeted to the plastid (Figure S6.4.1) and it is possible that this is in fact related to the absence of the gamma subunit.

Targeting sequences of kareniacean plastid proteins vary with their evolutionary origin

Next, we wished to test if there are differences in the plastid targeting pre-sequences of plastid-late and plastid-early proteins, by comparison to analogous protein regions from peridinin dinoflagellate and haptophyte references. The signal peptides of kareniacean plastid-targeted proteins were found to typically contain a central LACLAC motif and a terminal GHG motif directly preceding the cleavage site regardless of whether they were of plastid-late or plastid-early origin (Figure 4A-B), whereas these motifs do not occur in haptophytes nor peridinin dinoflagellates (Figure 4C-D). Comparisons of the unique three-letter motifs associated with the plastid-targeting sequences of each group revealed particularly strong enrichments in the LACLAC and GHG motif and its variants in kareniacean proteins of plastid-late origin, compared to proteins of plastid-early origin and haptophyte and dinoflagellate equivalents (Figure 4E), despite similar overall amino acid composition of signal peptides across all datasets (Figure S7). In contrast, limited similarities were found in the transit peptides of kareniacean plastid-targeted proteins, apart from the double-arginine motif immediately following the cleavage site and conserved proline at +15 and arginine at +22 after the cleavage sites (Figure 4F).

Global distribution of fucoxanthin dinoflagellates reveals Karlodinium as an outlier and a negative co-occurrence with their haptophyte progenitors

To provide biogeographical and ecological context for the interpretation of our transcriptomic and phylogenetic results we investigated the distribution of Karenia, Karlodinium, and Takayama in the Tara Oceans database using V9 18S metabarcoding (data available in Tables S5-8). Our analysis shows that Karlodinium clearly differs from the other two genera in its higher overall abundance and station occupancy and more cosmopolitan distribution, which is noticeable especially in the Indian and Arctic Oceans (Figure 5). Partial Least Square (PLS) analysis shows a set of environmental variables (salinity, silicate, iron) positively correlated with abundances of both Karenia and Takayma and also haptophytes as a whole, but at the same time negatively correlated to Karlodinium (Figure S8), further illustrating that the latter genus is quite distant from the rest in its biogeographical pattern. There was no correlation between any of the three studied genera themselves (Figure S9).

We further performed PLS analysis of the abundances of the three kareniacean genera and haptophytes, divided by family. We note an overall negative correlation to Chrysochromulinales, Phaeocystales, and Coccolithales, and positive correlation to Pavlovales (Figure 6), reflected by the overall preference of fucoxanthin dinoflagellates for temperate and tropical stations. We note that the negative association of present-day fucoxanthin dinoflagellates with Chrysochromulinales contrasts with the probable Chrysochromulinalean signal within their plastid proteomes.

Discussion

In this study, we harness novel and previously sequenced transcriptome data, customized in silico prediction, automated phylogenomics, and metagenomic analyses from Tara Oceans to reveal the proteomic composition and deeper taxonomic affinities of the fucoxanthin-containing plastids of the kareniacean dinoflagellates, a group of a special evolutionary importance as a tractable model system for studying the serial endosymbiotic acquisition of plastids. Thus, our results illuminate the mechanistics of a fundamental process that may underpin vast tracts of chloroplast evolution.

Using a combined phylogenetic matrix of 22 curated plastid-targeted proteins most closely related to haptophytes we were able to propose, to our knowledge for the first time, an origin for the fucoxanthin plastid origin with a specific haptophyte subgroup. Although the single-gene tree topologies vary greatly, often returning Kareniaceae as non-monophyletic, a plurality identify a probable origin within the Chrysochromulinales, and the refined combined phylogeny retrieves Kareniaceae as monophyletic and sister to Chrysochromulinales with maximum support. The non-negligible number of proteins exhibiting evolutionary affiliation with other groups / trees exhibiting conflicting topologies may be a result of either phylogenetic noise, or LGT from other haptophytes having facilitated the establishment of the fucoxanthin plastid in a taxonomically localized version of the "shopping bag model" [START_REF] Larkum | Shopping for plastids[END_REF], particularly if the kareniacean ancestor was specialized to predation on haptophyte prey. We note that the divergence date inferred between Chrysochromulinales and Prymnesiales within the haptophytes from molecular clock data, i.e., between approximately 150 and 300 million years ago [START_REF] Medlin | A molecular clock for coccolithophores and implications for selectivity of phytoplankton extinctions across the K/T boundary[END_REF][START_REF] Liu | A Time line of the Environmental Genetics of the Haptophytes[END_REF], is coherent with the probable divergence of Kareniaceae from other dinoflagellate groups (no more than 250 million years ago), and is certainly more plausible than an origin between the divergence of the Pavlovophytes and radiation of Prymnesiophyceae (> 500 million years ago) [START_REF] Parfrey | Estimating the timing of early eukaryotic diversification with multigene molecular clocks[END_REF][START_REF] Strassert | A molecular timescale for eukaryote evolution with implications for the origin of red algal-derived plastids[END_REF] as has been inferred from plastid-encoded gene phylogenies to demarcate the origin of the fucoxanthin plastid [START_REF] Kawachi | Rappemonads are haptophyte phytoplankton[END_REF].

Our phylogenetic results are further correlated with environmental abundances in present-day metagenomic data. Chrysochromulinales, the presumed donor group of the plastid and also largest portion of the sortable haptophyte-like genes, is most strongly anti-correlated to all three kareniaceans, while the only group with which they are consistently positively correlated, Pavlovales, is the one that had the most negligible genetic imprint on their plastid proteins (compare Figure 1B and Figure 6). Isochrysidales, the genetic imprint of which seems to be the second largest, are positively correlated only to Karenia, not Karlodinium and Takayama. This pattern might reflect an active predation avoidance that may have evolved in those haptophyte lineages that were ancestrally common prey and therefore also potential gene and plastid source for kareniaceans, while the very weak correlation of Pavlovales to kareniacean dinoflagellates suggests no or very little predator-prey interaction, reflected in the limited contribution of the Pavlovales to the fucoxanthin plastid proteome. Nonetheless, this result is not easy to interpret without more data regarding the current predator-prey intractions between kareniaceans and haptophytes and their specificity, e.g., whether this may point towards predator avoidance and/or adaptation for different niches or, conversely, efficient clearing of the prey population by the predators Alternatively, the different geographical distributions of the chrysochromulinalean and kareniacean lineages may reflect contrasting environmental preferences. In this case, the acquisition of the fucoxanthin plastid by the kareniacean dinoflagellate host may have been accompanied by a transition to a new ecological niche, i.e., into tropical and oligotrophic instead of polar and nutrient-rich waters [START_REF] Penot | Genomic and meta-genomic insights into the functions, diversity and global distribution of haptophyte algae[END_REF], with the associated physiological constraints directing its subsequent evolution. It is important to note in either case that the current biogeographical patterns of each lineage may not be at all indicative of the situation at time of the endosymbiotic event. The exact significance of the ecological interactions between the progenitors of Kareniaceae, and the haptophyte ancestors of their fucoxanthin plastid, may be best inferred via ancestral niche reconstruction for each lineage (as e.g., have been attempted by Moen and Morlon, 2014).

Strikingly, the plastid protein evolution suggested by our data -Karlodinium as sister to Karenia instead of Takayamadoes not copy the nuclear topology of the kareniacean lineage. We even note that the plastid-encoded gene topology produced does not necessarily suggest monophyly of the Kareniacean plastid genome. While we cannot exclude that this is a result of gene duplication and differential retention of paralogs [START_REF] Qiu | Ancient Gene Paralogy May Mislead Inference of Plastid Phylogeny[END_REF], limited phylogenetic signal, and/or independent horizontal acquisitions of plastid-targeted proteins by Takayama, our concatenated topology need not imply a truly singular evolutionary origin of the organelle. Takayama differs from other kareniaceans in the shape of the feeding apparatus and preferred prey size -it feeds on various other dinoflagellates including large species (over 60μm) [START_REF] Jeong | Mixotrophy in the phototrophic dinoflagellate Takayama helix (family Kareniaceae): Predator of diverse toxic and harmful dinoflagellates[END_REF], and may have consumed different prey than other fucoxanthin-containing lineages, which in turn may have differentially biased the potential sources of posterior LGT. At the same time, recent SSU and LSU phylogenies of Kareniaceae incorporate multiple species that do not possess a fucoxanthin plastid, that is RSD [START_REF] Hehenberger | A kleptoplastidic dinoflagellate and the tipping point between transient and fully integrated plastid endosymbiosis[END_REF], Gertia [START_REF] Takahashi | Gertia stigmatica gen. et sp. nov. (Kareniaceae, Dinophyceae), a New Marine Unarmored Dinoflagellate Possessing the Peridinin-type Chloroplast with an Eyespot[END_REF], and Shimiella [START_REF] Ok | Shimiella gen. nov. and Shimiella gracilenta sp. nov. (Dinophyceae, Kareniaceae), a Kleptoplastidic Dinoflagellate from Korean Waters and its Survival under Starvation[END_REF] as closer relatives to Karlodinium and Takayama to the consistent exclusion of Karenia. RSD and Gertia retain the ancestral perdinin plastid, in a residual and slightly reduced but photosynthetically active and peridinin-containing forms, respectively. Phylogenetic analyses of the RSD by us and other authors shows next to no haptophyte-like genes shared with the other three genera, strongly suggesting that it indeed never possessed a stable fucoxanthin plastid [START_REF] Hehenberger | A kleptoplastidic dinoflagellate and the tipping point between transient and fully integrated plastid endosymbiosis[END_REF]. We propose a scenario in which the fucoxanthin plastid of kareniaceans is indeed monophyletic, but was first acquired by the specific lineage leading to Karlodinium and Takayama after the divergence of the lineage(s) leading to the extant representatives with no or residual peridinin plastids. After this plastid was stabilized in the lineage leading to Karlodinium, it may have undergone an additional round of serial endosymbiosis spreading to the common ancestor of Karenia (Figure 7). We note that predator-prey relationships are not uncommon even between two toxin-producing dinoflagellates [START_REF] Jeong | Mixotrophy in the phototrophic dinoflagellate Takayama helix (family Kareniaceae): Predator of diverse toxic and harmful dinoflagellates[END_REF]. Conversely, the smaller size [START_REF] De Salas | Novel unarmored dinoflagellates from the toxigenic family Kareniaceae (Gymnodiniales): Five new species of Karlodinium and one new Takayama from the Australian sector of the Southern Ocean[END_REF] and cosmopolitan distribution (Figure 5) of Karlodinium might make it a potential prey for Karenia, although this awaits direct observation. Alternatively, the hypothetical additional serial endosymbiosis may have involved Takayama and a close relative of the chrysochromulinalean plastid donor of Karenia and Karlodinium.

The evolutionary patterns in the compiled plastid metabolic pathways and complexes provides insights into the specific roles of EGT, LGT and host-derived proteins in the early evolution of the fucoxanthin plastid. The preferential incorporation of plastid-late pathways into photosynthesis-associated pathways may reflect that these functions were not encoded in the kareniacean host and that the new plastid (or much less frequently LGT) were the only available sources. In contrast, the retention of dinoflagellatederived proteins in other pathways (e.g., isoprenoid, haem and amino acid biosynthesis) implies that the kareniacean ancestor retained these functions. We note that these functions are also often associated with protist lineages that have secondarily lost photosynthesis but retain vestigial plastids [START_REF] Hadariová | Reductive evolution of chloroplasts in non-photosynthetic plants, algae and protists[END_REF][START_REF] Dorrell | Principles of plastid reductive evolution illuminated by nonphotosynthetic chrysophytes[END_REF]. It has previously been suggested for the RSD that the peridinin plastid is retained in a non-photosynthetic form [START_REF] Hehenberger | A kleptoplastidic dinoflagellate and the tipping point between transient and fully integrated plastid endosymbiosis[END_REF], and such an organelle might be the donor of the plastid-early pathways now found in the fucoxanthin plastid. One interesting case illustrating this scenario is the plastid-early chlorophyllase, the only enzyme of this origin in the chlorophyll branch of tetrapyrrole metabolism, which might have been retained by a heterotrophic kareniacean ancestor for the purpose of phytol recycling from chlorophyll contained in its algal prey. Ultimately, however, the presence (or eventual fate) of the peridinin-containing plastid in fucoxanthin-containing lineages awaits structural confirmation via microscopy.

The predominantly plastid-late origins of plastid translation but apparent retention of ancestral RNA processing pathways (e.g., poly(U) tail addition and substitutional editing) in the fucoxanthin plastid is puzzling in this regard, insofar as the peridinin plastid only encodes core photosystem subunits, and a nonphotosynthetic plastid would be expected to lose its plastid genome [START_REF] Dorrell | Functional remodeling of RNA processing in replacement chloroplasts by pathways retained from their predecessors[END_REF][START_REF] Jackson | A tertiary plastid gains RNA editing in its new host[END_REF][START_REF] Cooney | Phylogenomics shows unique traits in Noctilucales are derived rather than ancestral[END_REF]. We note that RNA editing is widespread however in dinoflagellate mitochondrial and nuclear genomes [START_REF] Lin | Widespread and Extensive Editing of Mitochondrial mRNAS in Dinoflagellates[END_REF][START_REF] Liew | Condition-specific RNA editing in the coral symbiont Symbiodinium microadriaticum[END_REF], and also present in the green plastids of Lepidodinium that also secondarily replaced the peridinin-containing ones [START_REF] Matsuo | Comparative Plastid Genomics of Green-Colored Dinoflagellates Unveils Parallel Genome Compaction and RNA Editing[END_REF], and it remains to be determined if the fucoxanthin plastid RNA processing machinery is of relict peridinin plastid or non-plastidial host origin. It is also possible that there has been a direct niche competition between the peridinin and fucoxanthin plastid that may have coexisted in the same host for a period of time with possibly different selective pressure on retention of their respective proteins based on their interaction with plastid-encoded components, e.g., extrinsic photosystem subunits not assembling correctly with their intrinsic haptophyte-like counterparts. This would explain the strong bias towards plastid-late signal in photosynthetic machinery and ribosomes without the need for a period of heterotrophy with non-photosynthesizing plastid in the scenario, which could in turn make the abovementioned retention of peridinin-containing plastid in Gertia less baffling.

The general evolutionary division in the plastid metabolic map is not between plastid-late and plastidearly components but rather between parts strongly biased towards plastid-late and parts with no bias, assembled from both sources, likely in an evolutionarily neutral, patchwork-like way. This patchwork pattern does not only manifest at the level of one particular pathway being formed by enzymes of different evolutionary affiliations but often also at the level of one step in the pathway being carried out by homologs of different origin in different organisms. This division sometimes goes between the genera (e.g., plastid-late in Karenia, plastid-early in Karlodinium, consistent with the abovementioned overall higher proportion of plastid-late signal in the former; or Takayama appearing different from the rest) but sometimes even between individual species with no clear pattern. We propose that fucoxanthin plastid establishment may have been a relatively relaxed and gradual process during which peridinin-like and haptophyte-like genes may have both been present and competed for place in the nascent organelle (which was ultimately determined by neutral selection), not only during initial stages but also after the divergence of the genera and even species. We note that this phenomenon is also reflected in the partial plastid genomes assembled from Karenia and Karlodinium, which contain partially non-overlapping sets of genes that suggest independent post-endosymbiotic plastid genome reduction in each lineage [START_REF] Gabrielsen | Genome Evolution of a Tertiary Dinoflagellate Plastid[END_REF][START_REF] Richardson | Genome-Wide Transcript Profiling Reveals the Coevolution of Plastid Gene Sequences and Transcript Processing Pathways in the Fucoxanthin Dinoflagellate Karlodinium veneficum[END_REF][START_REF] Dorrell | Diversity of transcripts and transcript processing forms in plastids of the dinoflagellate alga Karenia mikimotoi[END_REF]. It remains to be understood why a higher proportion of plastid-late proteins are observed in Karenia than Karlodinium or Takayama, which may variously relate to a greater retention of plastid-early genes in the lineage leading to Karlodinium and Takayama, greater post-endosymbiotic reduction and relocation of plastid-encoded genes from the Karenia plastid to the nucleus, greater paralogizations of plastid-late genes, or potentially additional LGT of genes from haptophytes specifically into the Karenia lineage. Deeper sampling of fucoxanthin plastid genomes, and of the haptophyte lineages they predate, may provide clues into the mechanistic processes guiding their deep evolution.

From the diversity of signal sequences targeting proteins into fucoxanthin plastids, we consider possible structural similarities that may reveal their underlying evolutionary origin. The most striking feature of kareniacean signal peptides (both of plastid-late and plastid-early proteins) is their high degree of conservation, which is evident from the comparisons of sequence logos (Figure 4A-D) and the fact that they are highly similar to one another, suggesting either their shared origin or very strict evolutionary pressure on their sequence. At the same time, they are very dissimilar to those of either of their potential sources, peridinin dinoflagellates and haptophytes. If they were indeed inherited from one or the other, they evolved profoundly under very strong selection to the point of being more strictly conserved than their ancestors and untraceable. The other possibility is that they arose de novo during fucoxanthin plastid establishment, and their highly similar sequence reflects their common and possibly relatively recent origin and spread throughout the kareniacean genome. Previous studies have suggested the significance of transposable elements for the recruitment of targeting sequences to nascent nucleus-encoded and plastidtargeted proteins [START_REF] Burki | Intragenomic Spread of Plastid-Targeting Presequences in the Coccolithophore Emiliania huxleyi[END_REF], and the recent assembly of a draft Karenia brevis nuclear genome may allow us to assess to what extent transposons were involved in the transition of a transient kareniacean symbiont to an integrated organelle whose biogenesis is controlled by the host and its nuclear genome [START_REF] Nelson | Large-scale genome sequencing reveals the driving forces of viruses in microalgal evolution[END_REF].

Finally, we note that the basic metabarcoding analysis of the biogeographical distribution of Kareniaceae in Tara Oceans data is preliminary, particularly as it relies on analysis for the three genera due to the lack of species-level assignations of individual meta-barcodes. Meta-genome and meta-transcriptome abundances of plastid-late genes may enable a more precise enumeration of the environmental presence of the fucoxanthin plastid, particular by allowing the differentiation of fucoxanthin-containing Kareniaceae from peridinin-containing and non-photosynthetic relatives. Due to the almost completely translational regulation of the dinoflagellate nuclear genome [START_REF] Lidie | Gene expression in Florida red tide dinoflagellate Karenia brevis: Analysis of an expressed sequence tag library and development of DNA microarray[END_REF][START_REF] Carradec | A global ocean atlas of eukaryotic genes[END_REF], elucidating the precise physiological functions of individual fucoxanthin plastid proteins may however depend on more elaborate approaches, e.g., ribosome profiling of cultured species or environmental transcriptome data [START_REF] Bowazolo | Orchestrated translation specializes dinoflagellate metabolism three times per day[END_REF]. These eco-physiological insights may also be specific to each individual fucoxanthin dinoflagellate genus, particularly Takayama which seems to be notably separated from the rest in terms of its phylogenetic signal ratio and distribution among plastidial functions, as well as in its position in the phylogenies retrieved from plastid-targeted and plastid-coded genes; and Karlodinium, which represents an ecological outlier regarding abundance, stations occupancy, and correlation with environmental factors.

Conclusions

Seven in silico plastid proteomes of fucoxanthin plastid-bearing kareniaceans were retrieved from already available and newly generated transcriptomic data and phylogenetically analysed using large-scale automated tree building and sorting. The ratio between plastid-late and plastid-early phylogenetic signal differs between the three studied genera and its uneven distribution among functionally annotated plastid enzymes in individual lineages suggests that the serial plastid establishment and formation of its evolutionarily mosaic proteome was a relatively long and relaxed process with room for significant diversification. Phylogenomic analyses of the haptophyte-like sequences reveal Chrysochromulinales as the closest relatives and possible donor lineage of the fucoxanthin plastid for the first time but at the same time suggests that its evolutionary history inside Kareniaceae was not straightforward and may have involved extensive horizontal gene flow or even additional serial endosymbiosis. Additionally, we reconstructed a comprehensive composite metabolic map of the fucoxanthin plastid, investigated kareniacean plastid signal sequences with regard to their phylogenetic origin, and analysed the global distribution of the three kareniacean genera in regard to both abiotic and biotic factors. We emphasise the importance of considering micro-evolutionary variation in the establishment of plastids in the Kareniacean dinoflagellates, and across the broader algal tree of life.

Materials and methods

Reference database preparation

An in-house protein database was used for homology searches throughout this project. This database consisted of 155 transcriptomic and genomic datasets fetched from NCBI (https://www.ncbi.nlm.nih.gov), 1KP (https://db.cngb.org/onekp/; [START_REF] Leebens-Mack | One thousand plant transcriptomes and the phylogenomics of green plants[END_REF] and decontaminated MMETSP (Keeling et al., 2014) as described and in Dorrell et al., 2021: 30 for eubacteria, 10 for archaea, and 115 for eukaryotes with sampling spanning most of the diversity with focus on plastid-bearing groups (100 datasets) and particularly enriched in dinoflagellate and haptophyte sequences (37 and 34 datasets, respectively). More details and list of used datasets is available in Table S2).

Data acquisition and de novo transcriptome sequencing

The previously published peptide sequences for Karenia brevis, Karlodinium micrum (syn. veneficum) and Ross Sea Dinoflagellate (RSD) were downloaded from their respective public repositories (Keeling et al., 2014;[START_REF] Ryan | De novo assembly and characterization of the transcriptome of the toxic dinoflagellate Karenia brevis[END_REF][START_REF] Hehenberger | A kleptoplastidic dinoflagellate and the tipping point between transient and fully integrated plastid endosymbiosis[END_REF] and decontaminated as described above. In case of K. brevis, the four available assemblies from four clones were pooled together and treated as a single dataset with redundant sequences removed randomly.

Karenia mikimotoi RCC1513, Karenia papilionacea RCC6516 and Karlodinium sp. RCC3446 were obtained from Roscoff Culture Collection (https://roscoff-culture-collection.org), Takayama helix CCMP3082 was obtained from the Bigelow National Center for Marine Algae and Microbiota (https://ncma.bigelow.org), and Karlodinium armiger K0668 was obtained from the Norwegian Culture Collection of Algae (https://niva-cca.no). The cultures were grown in natural seawater (Horsey Mere seal colony, North Sea) supplemented with k/2 or enriched artificial seawater on a 12 hrs light (50µE): 12 hrs dark cycle at 19°C for 4 weeks, to late-exponential phase (1-2 million cells/ml). Cells were concentrated by centrifugation at 4000 rpm for 10 minutes, washed three times in sterile marine PBS (mPBS), and snap-frozen in liquid nitrogen. Total cellular RNA was extracted as previously described [START_REF] Dorrell | Functional remodeling of RNA processing in replacement chloroplasts by pathways retained from their predecessors[END_REF] using Trizol (Invivogen) and chloroform phase extraction, isopropanol precipitation, and resuspended in nuclease-free water (Qiagen). RNA integrity was confirmed by electrophoresis on ethidium bromide-stained agarose gels and quantified using a nanodrop photospectrometer. Two µg RNA was subsequently treated with 2U RNA-free DNAse (Promega) following the manufacturers' instructions, precipitated with isopropanol, resuspended in nuclease-free water and quality-checked and quantified again.

For each species, 1 µg DNase-treated RNA was polyA-tail selected, and strand-specific libraries were prepared with TruSeq Stranded mRNA Library Prep kit (Illumina). The resulting libraries were sequenced using a Novaseq flow cell (Fasteris) with 100 bp paired reads, followed by in-house base-calling and basic Q30 enumeration (https://www.fasteris.com).

The obtained paired-end reads were further quality controlled using the RNA-QC-chain toolkit [START_REF] Zhou | RNA-QC-chain: comprehensive and fast quality control for RNA-Seq data[END_REF] by filtering based on phred score with the threshold of 30; and GC content with the allowed range set to 42-60% based on the expected values for kareniacean dinoflagellates [START_REF] Lidie | Gene expression in Florida red tide dinoflagellate Karenia brevis: Analysis of an expressed sequence tag library and development of DNA microarray[END_REF], and by removing reads potentially representing rRNA contamination (default settings were used for this step).

Filtered reads were then assembled by Trinity [START_REF] Grabherr | Fulllength transcriptome assembly from RNA-Seq data without a reference genome[END_REF] with --NO_SEQTK and -trimmomatic options and the resulting assembly was selected for the longest isoforms by the respective script from the Trinity toolkit. The quality and completeness of the transcriptomic datasets were assessed by TrinityStats and BUSCO (eukaryota_odb10, n:255) [START_REF] Simão | BUSCO: Assessing genome assembly and annotation completeness with single-copy orthologs[END_REF]. The datasets were translated to proteins by TransDecoder (with default settings; https://github.com/TransDecoder/TransDecoder) and subsequently decontaminated by homology search using LAST [START_REF] Kiełbasa | Adaptive seeds tame genomic sequence comparison[END_REF] against the in-house database and removing sequences homologous to eubacterial, archaeal, or metazoan sequences with evalue of 1E-10 or lower. The translated datasets were used for further analyses alongside the previously published transcriptomes in their translated forms and are available at https://figshare.com/articles/dataset/full_protein_datasets_tar/21647771. The sequence data were deposited at NCBI under BioProject ID PRJNA788341.

Plastid signal prediction

SignalP 5.0 [START_REF] Almagro Armenteros | SignalP 5.0 improves signal peptide predictions using deep neural networks[END_REF] was used for prediction of N-terminal signal peptides as it was determined the version providing the most specific plastid prediction during preliminary optimization. Both original sequences and sequences cut to the first methionine were analysed to accommodate for potential translated UTRs or spliced leaders, and the results were pooled afterwards prioritizing the positive signal peptide prediction in case of different results. The obtained SignalP results were converted into a tabular format compatible with SignalP versions 3.0-4.1 for downstream applications with a custom script (SP5toSP4.py). For the second step predicting the rest of the bipartite targeting signal, a modified version of ASAFind [START_REF] Gruber | Plastid proteome prediction for diatoms and other algae with secondary plastids of the red lineage[END_REF][START_REF] Füssy | Subcellular Compartments Interplay for Carbon and Nitrogen Allocation in Chromera velia and Vitrella brassicaformis[END_REF] was used with a custom scoring matrix (modASAFind.py, Table S9) that reflects the differences in sequence surrounding the signal peptide cleavage site of kareniaceans (for instance, instead of the phenylalanine motif, multiple arginine residues are typically present). The matrix was prepared based on sequence logos prepared by Seq2Logo [START_REF] Thomsen | Seq2Logo: A method for construction and visualization of amino acid binding motifs and sequence profiles including sequence weighting, pseudo counts and two-sided representation of amino acid enrichment and depletion[END_REF]; Figure S10) from model datasets of Karenia brevis and Karlodinium micrum proteins determined as having exclusively plastidial, mitochondrial, nuclear, or endomembrane functions, e.g., photosynthetic core machinery proteins for plastidial functions, and identified from reciprocal BLAST against well-curated proteomic datasets from other organisms [START_REF] Butterfield | An analysis of dinoflagellate metabolism using EST data[END_REF][START_REF] Klinger | Comparative Genomic Analysis of Multi-Subunit Tethering Complexes Demonstrates an Ancient Pan-Eukaryotic Complement and Sculpting in Apicomplexa[END_REF][START_REF] Dorrell | Chimeric origins of ochrophytes and haptophytes revealed through an ancient plastid proteome[END_REF][START_REF] Bannerman | Multispecies reconstructions uncover widespread conservation, and lineage-specific elaborations in eukaryotic mRNA metabolism[END_REF][START_REF] Beauchemin | A proteomic portrait of dinoflagellate chromatin reveals abundant RNA-binding proteins[END_REF], the queries and retrieved model sequences are available in Tables S10-11. The modified script and custom scoring matrix were subsequently tested and optimized for sensitivity and specificity, and in the final version represented an improvement of 56.4 and 0.5 percentage points, respectively over unmodified ASAFind. The detection thresholds used with SignalP were also tested, with no apparent improvement in specificity/sensitivity tradeoffs over the default settings; as well as prediction by HECTAR and combination of PrediSI and ChloroP [START_REF] Gschloessl | HECTAR: A method to predict subcellular targeting in heterokonts[END_REF][START_REF] Dorrell | Principles of plastid reductive evolution illuminated by nonphotosynthetic chrysophytes[END_REF][START_REF] Ebenezer | Transcriptome, proteome and draft genome of Euglena gracilis[END_REF]Figure S11-12, Tables S12-16).

Additionally, we employed an alternative prediction, previously tested on the unrelated eukaryote Euglena gracilis combining PrediSI [START_REF] Hiller | PrediSi: Prediction of signal peptides and their cleavage positions[END_REF] and ChloroP [START_REF] Emanuelsson | ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites[END_REF] and proteins judged negative by modified ASAFind but positive by this approach were included in the predicted plastid proteomes as well albeit with prefix "SPTP-" denoting the presence of an alternative signal.

The presence of large numbers of highly similar sequences caused by very recent paralog duplication and/or alternative splicing is common in dinoflagellates. To reduce this complexity for phylogenetic analyses, all datasets were additionally clustered by cd-hit with an identity threshold of 0.9. This was performed after signal prediction to avoid accidentally discarding an isoform/paralog with predictable targeting signal in favor of one without it.

Phylogenetic analysis

Each of the in silico plastid proteome datasets was processed independently in order not to omit any variant signals in individual proteins. First, homologs were mined by LAST [START_REF] Kiełbasa | Adaptive seeds tame genomic sequence comparison[END_REF] search with threshold e-value 1E-10 against the in-house database plus all the fucoxanthin dinoflagellate transcriptomes. The best hit from all organisms in the in-house dataset were retained alongside the best five hits from each other fucoxanthin dinoflagellate library to account for potential paralogs in fucoxanthin dinoflagellate transcriptomes. Proteins with no hits whatsoever or with no homologs in any taxa other than dinoflagellates (Kareniaceae or other) were annotated as "lineage-specific" or "dinoflagellate-specific" respectively and their phylogeny was not investigated further. The remaining homolog sets were aligned by MAFFT (version 7.310, Mar 17 2017;[START_REF] Katoh | MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability[END_REF] with default settings and trimmed by TrimAl (version 1.4, Dec 17 2013;[START_REF] Capella-Gutierrez | trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses[END_REF] withgappyout parameter. The trimmed alignments were then filtered by a custom python script that discarded sequences comprising of more than 75% gaps and then rejected alignments shorter than 100 positions or containing less than 10 taxa. The satisfactory alignments were then used for maximum-likelihood tree construction by IQ-TREE (multicore version 2.0.5, May 15 2020; [START_REF] Minh | IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era[END_REF] with automatic model selection and 1000 ultrafast bootstraps. For graphical summary of this pipeline, see Figure S2. All trees are available at https://figshare.com/articles/dataset/all-automatically-built-trees_tar/21647768; in-house scripts used in the pipeline are available among supplementary files (scripts.tar); as well as the final predicted plastid protein datasets with their phylogenetic annotations (phylo-annotated_proteins_sets.tar).

TreeSorter, a freeware program written in Python (https://github.com/vanclcode/treesorter/) builds abstract tree structure from a tree file in nexus format, takes arguments defining sets of taxa and criteria based on those sets that determine what constitutes a valid subtree. Each tree is traversed and the highest bootstrap value of an edge (bipartition) that produces a subtree containing the taxon of interest and matching the defined criteria is reported. Criteria allow for further quantification of taxon sets, such that minimum and/or maximum number or proportion of taxa from a particular set per subtree. TreeSorter was used for large-scale analysis of the tree topologies and sorting and annotating the seed plastid proteins by their probable evolutionary origin into custom categories: haptophyte (plastid-late), dinoflagellate (plastidearly), alveolate (non-plastid), green (i.e., green algal, plant, or chlorarachniophyte; potential LGT or EGT), brown (i.e., ochrophyte; potential LGT or EGT), prokaryotic (likely LGT) as sequences of these evolutionary origins were noticed in these organisms in previous studies [START_REF] Nosenko | Chimeric plastid proteome in the Florida "red tide" dinoflagellate Karenia brevis[END_REF]Waller et al., 2006), and other/unresolved. In order to be assigned to one of the categories, the seed protein had to be either sister to or nested within a clade comprising exclusively members of said category and of at least 2 taxa. The bootstrap value of the highest-supported branch dividing such clade from the rest of the tree was then saved as the numerical score of this sorting result. In case the protein can be assigned to more than one category, the one with higher bootstrap was selected, if they have equal score, the protein is considered unresolved, with the exception of nested categories (such as dinoflagellates and alveolates) where the wider category is selected. Bootstrap scores of 75 were generally considered as the lower threshold in interpreting the evolutionary origins, however under specific circumstances (e.g. when the same protein was assigned the same origin with score ≥75 in other studied species) lower scoring results are reported as well.

The in silico plastid-targeted proteomes were automatically annotated by KAAS and major metabolic pathways were reconstructed, either using KEGG Mapper (https://www.genome.jp/kegg/mapper/reconstruct.html), or manually by targeted HMMER search for missing enzymes or enzymes without representation in KEGG database and/ or based on their conserved domains as detected by PfamScan [START_REF] Finn | Pfam: The protein families database[END_REF].

Phylogenomic analysis

A concatenated phylogenetic matrix for plastid proteins was prepared using 22 genes that passed our criteria of a) tree-based evidence of plastid-late origins, b) occurrence in at least 5 of the 7 organisms, and c) manually verified functional annotations and plastid-associated functions: 1-acyl-sn-glycerol-3phosphate acyltransferase, 3-hydroxyacyl-ACP dehydratase, 3-oxoacyl-ACP synthase, ACP, cysteinyl-tRNA synthetase, digalactosyldiacylglycerol synthase, glutaminyl-tRNA synthetase, haem oxygenase, chlorophyll a synthase, chlorophyllide b reductase, lycopene beta cyclase, magnesium chelatase subunit H, magnesium-protoporphyrin O-methyltransferase, protochlorophyllide reductase, PsbO, PsbP, PsbU, RP-L1, RP-L13, SecA, SufC, and SufD. Preliminary single-gene trees were manually checked to remove paralogs, very long branches, and likely cases of LGT, if present; these trees with the removed taxa highlighted, as well as the original and trimmed datasets, and final matrix are available as supplementary files (plastid_protein_concatenation_files.tar). The phylogenetic matrix for non-plastid proteins was prepared using the standard workflow of the PhyloFisher toolkit [START_REF] Tice | PhyloFisher: A phylogenomic package for resolving eukaryotic relationships[END_REF], with the manual step of paralog annotation by parasorter, and additional removal of sequences containing >66% of gaps; the matrix constructor statistics, input metadata, and the matrix itself available as supplementary files (phylofisher_files.tar). Both concatenated trees were then constructed by IQ-TREE with LG+C60+F model for the plastid protein matrix and posterior mean site frequency (PMSF) model (LG+C60+F+G with a guide tree constructed with C20) for the non-plastid matrix [START_REF] Wang | Modeling Site Heterogeneity with Posterior Mean Site Frequency Profiles Accelerates Accurate Phylogenomic Estimation[END_REF]. Additionally, a constrained tree search and subsequent approximately unbiased (AU) topology test were performed by IQ-TREE on the plastid protein matrix to verify the recovered inner topology of Kareniaceae.

Signal peptide analysis

Four experimental datasets for this analysis consisted of two sets of plastid-targeted plastid-early (264) and plastid-late (437) proteins with assigned KEGG annotation with uniquely plastidial function and two analogous sets selected from MMETSP transcriptomes for five peridinin dinoflagellates (358) and five haptophytes (272) (details in Table S17) based on presence of a) detectable signal peptide and b) the same KEGG annotations to produce comparable datasets without functional bias. Signal peptide regions of these proteins were determined by SignalP 5.0 (the region of approximately 25 positions immediately following the predicted cleavage site was considered as putative partial transit peptide region). The regions were aligned by ClustalW, manually curated to keep their conserved part and used to create sequence logos for each dataset using WebLogo (https://weblogo.berkeley.edu/logo.cgi; [START_REF] Crooks | WebLogo: A sequence logo generator[END_REF]. The overall amino-acid composition and number of occurrences of three-letter sequence motifs suggested by the logos in each dataset was also determined.

Meta-barcoding analysis of Kareniaceae distributions

To explore the distribution of the three Kareniaceae genera, the V9 18S rDNA metabarcoding data from Tara Oceans corresponding to the genera Karenia, Karlodinium and Takayama were extracted from [START_REF] Ibarbalz | Global Trends in Marine Plankton Diversity across Kingdoms of Life[END_REF] (available at https://zenodo.org/record/3768510#.Ye-xqpHMJzp). Overall, a total of 69 barcodes were extracted for Karenia, 76 for Karlodinium and 131 for Takayama. Their respective distributions (at least equal to three reads) in the eukaryotic-enriched size fractions (Pierella Karlusich et al., 2019) (0.8-5 µm;5-20 µm;20-180 µm and 180-2000 µm) and two depths (surface and deepchlorophyll maximum) were pooled together and plotted using the R package 'mapdata' v2.3.0 (http://cran.nexr.com/web/packages/mapdata/index.html). The occupancy was estimated as the percentage of stations in which their respective barcodes were identified. A correlation analysis was conducted on the pooled Kareniaceae size fractions and depths, including a total of 75 stations in which at least one of the organisms was retrieved, with the R package 'corrplot' (https://github.com/taiyun/corrplot; [START_REF] Wei | R package "corrplot": Visualization of a Correlation Matrix[END_REF]. Two final Partial Least Square analyses were implemented with the R package 'plsdepot' (https://github.com/gastonstat/plsdepot) to explore the correlation between: 1) kareniacean abundances and the abundance of the 5 haptophyte taxa Chrysochromulinaceae, Coccolithales, Isochrysidales, Pavlovophyceae and Phaeocystales (V9 18S rDNA metabarcoding data extracted as above); 2) global kareniacean and haptophyte (all haptophyte taxa pooled) abundances compared with a diverse set of environmental variables (median values of nutrient concentrations of iron, nitrate, silicate, phosphate; and oxygen, salinity, temperature, all extracted from the PANGAEA database; [START_REF] Ardyna | Environmental context of all samples from the Tara Oceans Expedition[END_REF][START_REF] Guidi | Environmental context of all samples from the Tara Oceans Expedition (2009-2013), about sensor data in the targeted environmental feature[END_REF][START_REF] Guidi | Environmental context of all samples from the Tara Oceans Expedition[END_REF]. Both the abundance and environmental data considered for the Partial Least Square analyses were computed only for the surface with all four size fraction abundances pooled. Table 1: Assembly statistics and assessment of completeness for the five new transcriptomes. Figure 3: Reconstruction of major metabolic pathways of plastids of the seven kareniaceans, adapted from KEGG [START_REF] Kanehisa | KEGG: Kyoto Encyclopedia of Genes and Genomes[END_REF]. Each rosette (described in the legend) relates to the homologues of a specific nucleus-encoded and plastid-targeted protein, with each circle within each rosette corresponding to a different species, and different colours corresponding to different evolutionary affiliations. Proteins encoded in plastid genomes are provided for the incomplete genome assembly for K. micrum RCC3446, transcriptome-based set for K. mikimotoi and several sequences for K. brevis. A detailed breakdown of the individual sequences with their predicted origins used for this reconstruction are available in Table S3. Figure 7: An evolutionary scenario involving an additional serial endosymbiosis between Karlodiniumlike donor and an ancestor of Karenia which could explain the apparent non-monophyly of the fucoxanthin plastid suggested the phylogenetic incongruence revealed by the placement of recently described lineages without the organelle [START_REF] Takahashi | Gertia stigmatica gen. et sp. nov. (Kareniaceae, Dinophyceae), a New Marine Unarmored Dinoflagellate Possessing the Peridinin-type Chloroplast with an Eyespot[END_REF][START_REF] Ok | Shimiella gen. nov. and Shimiella gracilenta sp. nov. (Dinophyceae, Kareniaceae), a Kleptoplastidic Dinoflagellate from Korean Waters and its Survival under Starvation[END_REF] and reconstruction based on plastid-late proteins (see Figure 2). Plastid membrane layers and ultrastructures are drawn based on current microscopic observations for each species.

Tables

Karenia mikimotoi

Figure 1 :

 1 Figure1: Ratios of phylogenetic signal of plastid proteins with detectable homologs: plastid-late (haptophyte-like; orange), plastid-early (dinoflagellate-like or dinoflagellate-specific; blue), ancestrally alveolate-like (purple), and other or unclear (grey) (A), and further breakdown of the plastid-late signal traceable to a concrete haptophyte family (Chrysochromulinales (dark blue), Isochrysidales (blue), Prymnesiales (light blue), Phaeocystales (green), Coccolithales (yellow), and Pavlovales (orange)) (B).

Figure 2 :

 2 Figure2: The eight kareniaceans in the pan-eukaryotic phylogenetic context as reconstructed by IQ-TREE based on matrix of 241 genes prepared using PhyloFisher toolkit (A) versus the plastidial phylogeny reconstructed based on 22 proteins of previously determined plastid-late origin (B); bootstrap support is expressed by the branch colour (black for ≥90%, dark grey for ≥75%, light grey for <75%). While RSD places sister to its kleptoplastid donor Phaeocystis antarctica, the three non-RSD kareniaceans form a monophyletic clade sister to Chrysochromulinales with maximum bootstrap support on all branches. Inside this clade, Karenia and Karlodinium cluster together to the exclusion of Takayama.

Figure 4 :

 4 Figure4: Sequence logos for aligned signal peptides of the four datasets of different evolutionary identity: haptophyte, peridinin dinoflagellate, plastid-late and plastid-early kareniacean (A-D), occurences of threeletter sequence motifs in signal peptides of each dataset, represented as a percentage of signal peptides in which at least one such motif occurs (E), and sequence logo of the signal peptide and partial transit peptide for kareniacean plastidial proteins regardless of their phylogenetic origin with red arrow denoting the signal peptide cleavage site (F).

Figure 5 :

 5 Figure5: Abundances of the three studied kareniacean genera in Tara Oceans stations based on ribosomal 18S SSU V9 metabarcoding -Karlodinium exhibits higher global abundance and more cosmopolitan distribution in comparison to Karenia and Takayama; for a correlation circle with individual environmental variables, see FigureS9; none of the genera display significant positive or negative correlation to one another (FigureS10).

Figure 6 :

 6 Figure 6: Correlation circle based on the Partial Least Square analysis of the Tara Oceans abundances (surface depth only) of the three studied kareniacean genera against those of different haptophyte families (colour-coding is the same as in Figure 1B) as observable variables; all three kareniaceans are negatively correlated to Chrysochromulinales, Coccolithales, and Phaeocystales but positively correlated to Pavlovales while only Karenia is positively correlated to Isochrysidales.

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Acknowledgements

RGD and ANV acknowledge supporting from a CNRS Momentum Fellowship awarded to RGD, 2019-2021, and an ANR JCJC ("PanArctica", ANR-21-CE02-0014) awarded to RGD, 2021-2022. RGD further acknowledges an ANR t-ERC ("ChloroMosaic"), awarded 2022. CB acknowledges funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (Diatomic; grant agreement No. 835067), the French Government 'Investissements d'Avenir' programmes MEMO LIFE (ANR-10-LABX-54) and PSL* Research University (ANR-1253 11-IDEX-0001-02), and from the Agence Nationale de la Recherche (BrownCut; grant agreement ANR-19-CE20-0020). The authors thank Elisabeth Hehenberger for sharing translated and decontaminated RSD data and consultation regarding their analysis, Lukáš Novák for consultation regarding phylogenomics, and Emmanuel Alastra for aid in media substrate preparation. This article is contribution [will be provided at proof stage] to Tara Oceans.