Ryoma Kamikawa
email: kamikawa.ryoma.7v@kyoto-u.ac.jp

Takako Mochizuki

Mika Sakamoto

Yasuhiro Tanizawa

Takuro Nakayama

Ryo Onuma

Ugo Cenci

Daniel Moog

Samuel Speak

Krisztina Sarkozi

Andrew ||

Toseland

Cock Van Oosterhout

Kaori Oyama

Misako Kato

Keitaro Kume

Motoki Kayama

Tomonori Azuma

Ken-Ichiro Ishii

Hideaki Miyashita

Bernard Henrissat

Vincent Lombard

Joe Win

Sophien Kamoun

Yuichiro Kashiyama

Shigeki Mayama

Shin-Ya Miyagishima

Goro Tanifuji

Thomas Mock
email: t.mock@uea.ac.uk

Yasukazu Nakamura

Genome evolution of a nonparasitic secondary heterotroph, the diatom Nitzschia putrida

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

The loss of photosynthesis in photoautotrophs is successful if compensated by a competitive advantage arising from the availability of an extracellular energy source. Hence, many secondary heterotrophs evolve as parasites [START_REF] Freesec | Parasitism finds many solutions to the same problems in red algae (Florideophyceae, Rhodophyta)[END_REF][START_REF] Hadariová | Reductive evolution of chloroplasts in nonphotosynthetic plants, algae and protists[END_REF][START_REF] Janouškovec | Apicomplexan-like parasites are polyphyletic and widely but selectively dependent on cryptic plastid organelles[END_REF], relying on sufficient resources provided by their hosts. Well-studied examples are the Apicomplexa [e.g., [START_REF] Kissinger | The Plasmodium genome database[END_REF]], which have lost photosynthesis secondarily. However, the loss of photosynthesis can also lead to free-living secondary heterotrophs, which are as common as parasites [START_REF] Hadariová | Reductive evolution of chloroplasts in nonphotosynthetic plants, algae and protists[END_REF][START_REF] Kamikawa | Multiple losses of photosynthesis in Nitzschia (Bacillariophyceae)[END_REF][START_REF] Dorrell | Principles of plastid reductive evolution illuminated by nonphotosynthetic chrysophytes[END_REF][START_REF] Kayama | A non-photosynthetic green alga illuminates the reductive evolution of plastid electron transport systems[END_REF][START_REF] Kayama | Highly reduced plastid genomes of the non-photosynthetic dictyochophyceans Pteridomonas spp. (Ochrophyta, SAR) are retained for tRNA-Glu-based organellar heme biosynthesis[END_REF]. Despite their significance, our knowledge about the evolution of free-living secondary heterotrophs is very limited, and we therefore lack insights into evolutionary processes required for them to thrive without photosynthesis and independently of a resource-providing host. Given that a parasitic lifestyle accelerates the rate of evolution (cf. Red Queen hypothesis) [START_REF] Van Valen | Molecular evolution as predicted by natural selection[END_REF] and of loss of conserved orthologous genes [e.g., [START_REF] Sun | Large-scale gene losses underlie the genome evolution of parasitic plant Cuscuta australis[END_REF]], the genome analysis of a nonparasitic secondary heterotroph can provide insights uncompromised by parasite-specific adaptations. Hence, the diatom Nitzschia putrida, isolated from mangrove estuaries, is the ideal model to test these hypotheses because it is an example of a free-living secondary heterotroph [START_REF] Kamikawa | Multiple losses of photosynthesis in Nitzschia (Bacillariophyceae)[END_REF][START_REF] Li | Four new apochlorotic diatoms[END_REF] within the diverse group of largely photoautotrophic diatoms [START_REF] Field | Primary production of the biosphere: Integrating terrestrial and oceanic components[END_REF][START_REF] Mann | The species concept in diatoms[END_REF]. As several genomes of the latter have recently become available including close phylogenetic relatives [START_REF] Armbrust | 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] Mock | Evolutionary genomics of the cold-adapted diatom Fragilariopsis cylindrus[END_REF], a genome-based comparative metabolic reconstruction of N. putrida promises to reveal fresh insights into what is required to thrive as a free-living secondary heterotroph. Thus, here, we have analyzed the draft genome sequence of N. putrida, which provides insight into evolutionary processes underpinning lifestyle shifts from photoautotrophy to free-living heterotrophy in the context of a coastal surface ocean ecosystem.

RESULTS

Genome assembly

K-mer-based GenomeScope analysis [START_REF] Vurture | GenomeScope: Fast reference-free genome profiling from short reads[END_REF] with 150-base pair (bp)long Illumina short reads suggested the genome of N. putrida (Fig. 1A) to be diploid (fig. S1A). To provide a high-quality genome with longrange contiguity, PacBio sequencing (RSII platform) was performed, resulting in ≥40-fold coverage. Because of the confirmed diploid nature of the N. putrida genome, we have applied the Falcon assembler and Falcon_unzip version 0.5 [START_REF] Chin | Phased diploid genome assembly with single-molecule real-time sequencing[END_REF] to provide a first draft genome of 2 of 12 this species. On the basis of this assembly, we estimated a genome size of 35 Mbp, including 87 scaffolds with an N50 of 860.9 kbp. The longest scaffold was 3.8 Mbp. The heterozygous regions of the genome (alternate contigs) estimated by the Falcon assembler resulted in 12 Mbp, with an N50 of 121 kbp (table S1). The Falcon assembly was error-corrected and polished by approximately 150-fold coverage of Illumina short reads, which were subsequently used for generating the final assembly with Pilon 1.2.2 [START_REF] Walker | Pilon: An integrated tool for comprehensive microbial variant detection and genome assembly improvement[END_REF] including manual curation.

According to the k-mer-assessed diploid nature of the N. putrida genome, the read coverage of the homozygous regions is approximately twofold higher than the read coverage for the heterozygous regions, suggesting the presence of diverged alleles as previously identified in the genome of the photoautotroph diatom Fragilariopsis cylindrus (fig. S1, A andB). Thus, most of the diverged allelic variants can be found in the heterozygous regions characterized by the presence of alternate contigs, whereas the regions with no corresponding alternate contigs are homozygous (fig. S1B). On the basis of the analysis with Braker2 version 2.0.3 [START_REF] Hoff | BRAKER1: Unsupervised RNA-Seq-based genome annotation with GeneMark-ET and AUGUSTUS[END_REF], the Nitzschia genome comprises 15,003 and 5767 inferred protein-coding loci on the primary and alternate contigs, respectively (table S1). Almost 40% of loci in the genome of N. putrida appear to be characterized by diverged alleles. A BUSCOv3 analysis [START_REF] Waterhouse | BUSCO applications from quality assessments to gene prediction and phylogenomics[END_REF] revealed the genome to be complete at a level of 90.1% based on the haploid set of genes.

The loss of photosynthesis

The haploid set of genes was used to reconstruct the nuclear-encoded plastid proteome of N. putrida and therefore to reveal the extent of gene loss including key genes of photosynthesis. A comparative analysis of the N. putrida plastome [START_REF] Gruber | Plastid proteome prediction for diatoms and other algae with secondary plastids of the red lineage[END_REF] with its photosynthetic counterparts revealed that more than 50% of nuclear encoded plastid proteins have been lost (Fig. 1B). More than 500 orthogroups (OrthoFinder) [START_REF] Emms | OrthoFinder: Solving fundamental biases in whole genome comparisons dramatically improves orthogroup inference accuracy[END_REF] of nuclear-encoded plastid proteins, which are usually shared between photosynthetic diatoms [START_REF] Gruber | Plastid proteome prediction for diatoms and other algae with secondary plastids of the red lineage[END_REF], are missing in the predicted plastid proteome of N. putrida (Fig. 1C). The missing part of the plastid proteome included genes encoding for proteins of lightharvesting antenna including fucoxanthin-chlorophyll a/c protein (fcp), photosystem II and I (e.g., psbA, psbC, psbO, psaA, psaB, and psaD), the cytochrome b6/f complex (e.g., petA), and carbon fixation (e.g., rbcS and rbcL) in addition to genes of the Calvin cycle [START_REF] Gruber | Plastid proteome prediction for diatoms and other algae with secondary plastids of the red lineage[END_REF], respectively. Data of two photosynthetic diatoms P. tricornutum and T. pseudonana are derived from the previous study [START_REF] Gruber | Plastid proteome prediction for diatoms and other algae with secondary plastids of the red lineage[END_REF]. (C) Unique and shared plastid-targeted orthogroups. Highlighted in red is the orthogroup exclusively shared by the two photosynthetic diatoms. (D) Predicted metabolic map of the nonphotosynthetic plastid. Representative pathways found in photosynthetic diatom species are shown. Green and light gray arrows show the presence and absence of the responsible protein sequences for the reactions in the genome, respectively. Amino acids are highlighted in red. Abbreviations are described in the Supplementary Materials.

S C I E N C E A D V A N C E S | R E S E A R C H A R T I C L E 3 of 12
[e.g., phosphoribulokinase (prk)]. Furthermore, a substantial number of key genes were missing for the biosynthesis of chlorophyll, carotenoids, and plastoquinones (fig. S1C).

Despite the loss of some of these key photosynthesis genes, there is still a substantial number of genes left encoding common plastid metabolic pathways as known from photosynthetic diatoms, including the generation of adenosine 5′-triphosphate (ATP) by adenosine triphosphatase (ATPase) subunits, which are encoded both in the nuclear and plastid genomes [START_REF] Kamikawa | Proposal of a twin arginine translocator system-mediated constraint against loss of ATP synthase genes from nonphotosynthetic plastid genomes[END_REF]. Almost all genes encoding for plastid enzymes to synthesize essential amino acids are still encoded in the nuclear genome of N. putrida. Furthermore, all genes of the heme pathway have been found, and N. putrida appears to be able to synthesize riboflavin. The presence of plastid-targeted transporters [START_REF] Moog | Substrate specificity of plastid phosphate transporters in a non-photosynthetic diatom and its implication in evolution of red alga-derived complex plastids[END_REF] enables the transport of phosphoenolpyruvate, 3-phosphoglycerate, and dihydroxyacetone-phosphate across the plastid membranes. In addition, our genome-based reconstruction of plastid metabolism identified the biosynthesis pathway for lipids and the ornithine cycle in N. putrida (Fig. 1D and fig. S2). The latter has been reported neither in previous transcriptome-based studies with this species [START_REF] Kamikawa | A non-photosynthetic diatom reveals early steps of reductive evolution in plastids[END_REF] nor in any other secondary heterotroph ([.g., [START_REF] Dorrell | Principles of plastid reductive evolution illuminated by nonphotosynthetic chrysophytes[END_REF][START_REF] Kayama | A non-photosynthetic green alga illuminates the reductive evolution of plastid electron transport systems[END_REF]]. As N. putrida has an osmotrophic lifestyle, it relies on dissolved organic matter and nutrients. Thus, the biosynthesis of a variety of metabolic compounds supports the osmotrophic lifestyle, lacking abilities to prey or to parasitize on other organisms.

Communication between organelles and light-dependent gene expression

The lack of CO 2 fixation in plastids of N. putrida-which reduces the amount of amino acids, lipids, and other metabolites to be synthesized-appears to be partially compensated for by the remodeling of metabolic interactions with mitochondria and peroxisomes and by retaining active recycling of nitrogen (Figs. 1 and2

and figs. S3 and S4

). It appears that the nonphotosynthetic plastid of N. putrida still exchanges glutamine and ornithine, both of which are important intermediates of the ornithine cycle. All genes for the ornithine-urea cycle have been retained in the N. putrida genome. The ornithineurea cycle is indispensable for nitrogen recycling in photosynthetic diatoms [START_REF] Allen | Evolution and metabolic significance of the urea cycle in photosynthetic diatoms[END_REF][START_REF] Smith | Evolution and regulation of nitrogen flux through compartmentalized metabolic networks in a marine diatom[END_REF], and even after the loss of photosynthesis, nitrogen recycling appears to be essential in N. putrida (Fig. 2A) due to its osmotrophic lifestyle. Usually, the ornithine-urea cycle is tightly linked with tricarboxylic acid cycle and/or photorespiration in photosynthetic diatoms [START_REF] Allen | Evolution and metabolic significance of the urea cycle in photosynthetic diatoms[END_REF][START_REF] Smith | Evolution and regulation of nitrogen flux through compartmentalized metabolic networks in a marine diatom[END_REF]. However, N. putrida is not likely to perform photorespiration (Fig. 2A). The metabolic exchange with the peroxisome through glycolate likely has ceased as phosphoglycolate phosphatase and peroxisomal glycolate oxidase are missing. Thus, photorespiration is unlikely to take place in nonphotosynthetic plastids of N. putrid due to the lack of ribulose 1,5-bisphosphate carboxylase/oxygenase and other key enzymes of the Calvin cycle (Fig. 1). Nevertheless, peroxisomes still appear to play a role in N. putrida for the production of malate or glyoxylate, which feed into respiratory pathways of the mitochondria to support ATP and NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) production (Fig. 2A).

Light in photosynthetic organisms not only plays a substantial role for photosynthesis generating ATP and NADPH but also regulates cell division, diel cycles, and different signaling processes unlike in many heterotrophic organisms [START_REF] Ashworth | Genome-wide diel growth state transitions in the diatom Thalassiosira pseudonana[END_REF][START_REF] Chauton | Gene regulation of carbon fixation, storage, and utilization in the diatom Phaeodactylum tricornutum acclimated to light/dark cycles[END_REF][START_REF] Smith | Correction: Transcriptional orchestration of the global cellular response of a model pennate diatom to diel light cycling under iron limitation[END_REF]. As a consequence, we identified the remaining photoreceptors and cell-cycle regulators such as cyclins and cyclin-dependent kinases [START_REF] Huysman | Genome-wide analysis of the diatom cell cycle unveils a novel type of cyclins involved in environmental signaling[END_REF]. Although they were still encoded and expressed in the genome of N. putrida (Fig. 2B and fig. S5, A andB), we were unable to identify a diel cycle in cell division (Fig. 2C). This suggests that these cel-cycle regulators potentially have neo/subfunctionalized and therefore have a different regulatory role in N. putrida unrelated to the diel cycle. The loss of the transcription factor bHLH-1a (RITMO1), which has been identified as a master regulator of diel periodicity [START_REF] Annunziata | bHLH-PAS protein RITMO1 regulates diel biological rhythms in the marine diatom Phaeodactylum tricornutum[END_REF], corroborates our finding that N. putrida has lost the ability to perform diel cycles. In addition, most of the other photoreceptors known from photosynthetic diatoms have also been lost (Fig. 2B) such as the blue light sensing aureochromes 1a/b, both of which are transcription factors responsible for photoacclimation [START_REF] Kroth | An update on aureochromes: Phylogeny-mechanismfunction[END_REF]. Despite the lack of lightdependent cell-cycle regulation, a few remaining photoreceptors were identified including bHLH1b_PAS, aureochrome 1c, and cryptochrome-DASH/CPF2 (Fig. 2B) [START_REF] Ashworth | Genome-wide diel growth state transitions in the diatom Thalassiosira pseudonana[END_REF][START_REF] Coesel | Diatom PtCPF1 is a new cryptochrome/photolyase family member with DNA repair and transcription regulation activity[END_REF]. Basic ZIP [basic leucine zipper proteins (bZIP)] transcription factors having potentially light-sensitive Per-Arnt-Sim (PAS) domains (bZIP-PAS) [START_REF] Fortunato | Diatom phytochromes reveal the existence of far-red-light-based sensing in the ocean[END_REF] were also identified in the N. putrida genome such as homologs to bZIP6 and bZIP7 of Phaeodactylum tricornutum [START_REF] Rayko | Transcription factor families inferred from genome sequences of photosynthetic stramenopiles[END_REF]. The latter homolog has been duplicated and diversified in N. putrida (fig. S5C). The presence of bZIP-PAS protein in a heterotrophic eukaryote is not unprecedented as some oomycetes, nonphotosynthetic parasites, have been reported to also encode them in their genomes [e.g., [START_REF] Kong | The basic leucine zipper transcription factor PlBZP32 associated with the oxidative stress response is critical for pathogenicity of the lychee downy blight oomycete Peronophythora litchi[END_REF]]. Although their role in regulating gene expression remains to be investigated in N. putrida, light still appears to influence the expression of some genes in this heterotrophic species. Comparative transcriptome analyses every 4 hours during a shift from a light phase to darkness (Fig. 2D) revealed eight clusters characterized by different expression patterns. Furthermore, there was no cluster explicitly representing the light-dependent gene expression patterns as seen in photosynthetic algae [e.g., [START_REF] Ashworth | Genome-wide diel growth state transitions in the diatom Thalassiosira pseudonana[END_REF][START_REF] Fujiwara | Relationship between cell cycle and diel transcriptomic changes in metabolism in a unicellular red alga[END_REF]]. However, one of the clusters contained genes only expressed in the mid-light phase: cluster 7 containing 90 genes (0.6% total). Forty four of them were genes with known functional domains based on a KOG (EuKaryotic Orthologous Groups) analysis, and 21 of them were encoding proteins for substrate import and carbon metabolism (fig. S5D). However, the photoreceptor homologs above-bHLH1b_PAS, aureochrome 1c, and cryptochrome-DASH/CPF2-were not part of this cluster, and there was no explicit trend in their gene expression patterns with respect to changes between light and dark conditions.

The genetic toolkit for the evolution of nonparasitic secondary heterotrophy

Despite the loss of many nuclear genes and their families, the genome size of N. putrida is not significantly different to photosynthetic relatives such as F. cylindrus and P. tricornutum and the more distantly related diatom Thalassiosira pseudonana (table S1). This is distinct from evolutionary trends observed in parasitic eukaryotes that have lost photosynthesis as they have smaller genomes encoding smaller gene families compared to their photosynthetic relatives (fig. S6). By comparing KOGs of paralogous proteins, there was no significant difference in the number of unique KOG IDs between these four diatom species (fig. S7, A andB). However, when we compared the number of paralogous proteins assigned to each KOG ID, there were several KOG categories for which N. putrida had a higher number of paralogous proteins compared to the other diatom species: nucleotide transport (F), transcription (K), signal transduction (T), intracellular trafficking, secretion, vesicular transport (U), and cytoskeleton (Z) (fig. S7C). Even after normalization by total gene numbers, nucleotide transport (F), signal transduction (T), Cluster 8

h 5 h 9 h 13 h 17 h 2h 25 h h 5 h 9 h 13 h 17 h 2h 25 h

Replicate A R eplicate B and cytoskeleton (Z) genes were more abundant in the N. putrida genome (fig. S8A). This observation was corroborated by N. putridaspecific enrichment of Pfam domains such as adenylate/guanylate cyclase and cyclic nucleotide esterase, leucine-rich repeat (LRR), and glycosyl/galactosyl transferase domains (fig. S8B).

A microbial heterotroph acquires nutrients either by phagotrophy, the preferred nutrition of many parasites, or by osmotrophy. The latter requires uptake of dissolved organic compounds by osmosis as realized by bacteria and fungi, for instance [START_REF] Richards | Horizontal gene transfer in osmotrophs: Playing with public goods[END_REF]41). As N. putrida grows well under axenic conditions [START_REF] Kamikawa | Multiple losses of photosynthesis in Nitzschia (Bacillariophyceae)[END_REF][START_REF] Ishii | Growth characterization of non-photosynthetic diatoms, Nitzschia spp., inhabiting estuarine mangrove forests of Ishigaki Island, Japan[END_REF], it is likely an osmotroph, dependent on the uptake of dissolved organic compounds across the silicified cell wall and the plasma membrane. As realized by osmotrophic fungi, N. putrida may even be able to degrade higher-molecular weight compounds extracellularly to be subsequently taken up as individual molecules by specific transporters or even osmosis [START_REF] Richards | Horizontal gene transfer in osmotrophs: Playing with public goods[END_REF]41). Thus, it is likely that cell wall, membrane, and secreted proteins were diversified in N. putrida compared to photosynthetic diatoms to facilitate osmotrophy.

To address this hypothesis, we analyzed the enrichment of paralog proteins and differences in nutrient transporters involved in the uptake of dissolved organic compounds such as solute carriers. A comparison to photosynthetic diatoms and parasitic nonphotosynthetic algal species [Prototheca and Helicosporidium (green algae), and the apicomplexans Plasmodium and Toxoplasma] has revealed that N. putrida has a unique composition of genes encoding transporters, which is therefore different to photosynthetic algae and parasitic nonphotosynthetic algal species (Fig. 3, A to C). For instance, the number of genes encoding silicon transporters (SITs), solute symporters, and the resistance-nodulation-cell division superfamily was more than twice as abundant in N. putrida compared to photosynthetic diatom species (Fig. 3D and fig. S9A). However, in contrast to the difference between N. putrida and photosynthetic diatom species, there is no enrichment of particular transporters in parasitic algal species when compared to their photosynthetic relatives (fig. S9, B andC).

Expansion of those gene families may, at least partly, have been achieved by recent tandem duplications (Fig. 3E). To gain insight into when the expansion had occurred, we performed a coalescence analysis, which revealed that SITs in N. putrida began to expand around 3.3 million years (Ma) ago [1.2 to 6.6, 95% confidence interval (CI)], while divergence from another nonphotosynthetic diatom N. alba is estimated to have occurred around 6.67 Ma ago (2.5 to 11.5, 95% CI; fig. S10). The split between F. cylindrus and P. multiseries, which was used to date the tree, was estimated at 9.7 Ma ago (7.6 to 11.6, 95% CI).

Thus, the recent expansion of SITs suggests neo/subfunctionalization of the gene family in response to the change in lifestyle. The divergence rate of SIT genes was much larger than that of control genes (e.g., myosin), indicating that SIT diversification might have contributed to the adaptation of the heterotrophic lifestyle. In support of this hypothesis, we detected several sites under positive selection in different members of the SIT family (table S2), which implies that the evolution of those genes may have been driven by diversifying selection.

The solute sodium symporters are estimated to have diverged around 7.5 Ma ago (3.8 to 11.1, 95% CI), markedly earlier than the SIT gene family. Although the divergence rate is also larger than that of control genes (fig. S10), we did not find evidence of diversifying selection in this gene family. The differences between these two families of transporters suggest that their expansion might have occurred in a stepwise manner and driven by different evolutionary forces.

Furthermore, although the overall carbohydrate-active enzyme (CAZyme) family composition of Nitzschia was not different from that of photosynthetic diatoms (fig. S11), families encoding -glycoside hydrolase (GH8), laminarinase (GH16_3), pectinase (GH28), -glucanase (GH72), -mannan hydrolyzing enzymes (GH99), and -1,2-glucan hydrolytic enzymes (GH114) were enriched in N. putrida compared to photosynthetic species (Fig. 3F). Expansion of these families might, at least partly, have been achieved by recent tandem duplications (Fig. 3G), suggesting an important role of these genes for the heterotrophic lifestyle of N. putrida. Notably, more than one-third of proteins assigned to the above six CAZyme families are predicted to be secreted in N. putrida (see below). The CAZyme compositions suggest that N. putrida might be able to degrade extracellular polysaccharides such as ß-1,3 glucans (e.g., lichenin, paramylon, callose, and laminarin), starches, -1,2-glucans, pectin, and -mannan. As N. putrida has been isolated from disintegrating mangrove leaves in a paddle [START_REF] Kamikawa | Multiple losses of photosynthesis in Nitzschia (Bacillariophyceae)[END_REF][START_REF] Ishii | Growth characterization of non-photosynthetic diatoms, Nitzschia spp., inhabiting estuarine mangrove forests of Ishigaki Island, Japan[END_REF], this species might play a role in degrading dead leaves and therefore facilitating carbon recycling in mangroves. To gain first insight into how transcription of CAZyme genes is regulated by different carbon sources, we performed comparative transcriptome analyses with starved N. putrida cells in comparison to cells growing on glucose and starch. However, we found that only a limited number of genes encoding CAZymes were differentially expressed (table S3). About half of these genes were upregulated in response specifically to starch as a carbon source, while only one CAZyme gene was up-regulated in response to glucose (table S3). This observation suggests that most of the CAZymes in N. putrida are not for the utilization of glucose and only very few for starch utilization. Arguably, providing a very limited set of organic substrates does not reflect the complexity of organic carbon provided by disintegrating leaves in a mangrove ecosystem. Hence, this might be the main reason for the limited transcriptome response observed in our experiments.

The predicted secretome of the nonparasitic, free-living secondary heterotroph N. putrida

Given that the secretome plays an important role for substrate degradation and subsequent uptake of low-molecular weight compounds in osmotrophs [START_REF] Richards | Horizontal gene transfer in osmotrophs: Playing with public goods[END_REF], we conducted a comparative analysis to predict secreted proteins of N. putrida in silico by identifying proteins with N-terminal signal peptides and a lack of transmembrane domains. The resulting proteins were clustered using TribeMCL [START_REF] Enright | An efficient algorithm for large-scale detection of protein families[END_REF], and plastid-and lysosome-localized proteins were subsequently removed using ASAFind according to their characteristic targeting motifs [START_REF] Gruber | Plastid proteome prediction for diatoms and other algae with secondary plastids of the red lineage[END_REF] and Pfam domains. The number of putatively secreted proteins is 978, 998, 596, and 718 in N. putrida, F. cylindrus, P. tricornutum, and T. pseudonana, respectively, which corresponds to between 5 and 7% of the total number of genes in their genomes (fig. S12A). Nevertheless, there were significant differences when we compared the diversity of proteins between these four diatom species (Fig. 4, A and B); N. putrida, on average, had a significantly higher number of proteins per tribe than any of the other diatom species (two-sided Wilcoxon signed-rank test; P < 0.01; Fig. 4C). In particular, proteins involved in heterotrophy such as organic matter degradation/modification including CAZymes and peptidases were more abundant in N. putrida

N it z s c h ia F r a g il a r io p s is P h a e o d a c ty lu m T h a la s s io s ir a P la s m o d iu m T o x o p la s m a V it r e ll a C h r o m e r a H e li c o s p o r id iu m P r o to th e c a A u x e n o c h lo r e ll a C h lo r e ll a C o c c o m y x a

The number of transporters involved in a particular transporter family 101 in T. pseudonana; fig. S12A). This is in contrast to parasitic green algae because their predicted secretomes are smaller than those of their photosynthetic relatives and show no explicit enrichment of secretome proteins per tribe (Fig. 4, D to F).

* GH79 GH137 GH7 GH158 GH63 GH9 GH43 GH107 GH125 GH31 GH78 GH26 GH55 GH10 GH1 GH30 GH72 GH36 GH161 GH18 GH19 GH29 GH28 GH3 GH20 GH81 GH47 GH2 GH38 GH99 GH5 GH16 GH110 GH35 GH89 GH116 GH128 GH17 GH27 GH8
S C I E N C E A D V A N C E S | R E S E A R C H A R T I C L E 7 of 12 LogFC -2 -1 0 1 2 1 2 3 4 1 h

h 9 h 13 h 17 h 21 h 2h 1 h h 9 h 13 h 17 h 21 h 2h

Replicate

N it z s c h ia F r a g il a r io p s is P h a e o d a c ty lu m T h a la s s io s ir a N it z s c h ia F r a g il a r io p s is P h a e o d a c ty lu m T h a la s s io s ir a N it z s c h ia F r a g il a r io p s is P h a e o d a c ty lu m T h a la s s io

The most common secreted proteins in N. putrida are LRRcontaining proteins (fig. S12B), many of which contain additional domains such as tegument and glycoprotein domains, suggesting an increased functional diversity (fig. S13). Only very few LRR-containing proteins were identified in the predicted secretomes of the photosynthetic diatoms, indicating that signal peptide-dependent secretion of abundant and diverse LRR-containing proteins may be an essential requirement in this secondary heterotroph, such as for environmental signaling [START_REF] Belkhadir | The growth-defense pivot: Crisis management in plants mediated by LRR-RK surface receptors[END_REF]. In addition to LRR-containing proteins, the top 10 most enriched proteins in N. putrida were von Willebrand factor type D (VWFD) proteins involved in adhesion or clotting, two types of endopeptidases, trypsin and leishmanolysin (cell-surface peptidase of the human parasite Leishmania), intradiol ring-cleavage dioxygenase protein for degradation of aromatic compounds, methyltransferase, and four proteins with unknown function (fig. S12B). LRR-containing proteins and VWFDs might play important roles in N. putrida for attaching to disintegrating mangrove leaves [START_REF] Kamikawa | Multiple losses of photosynthesis in Nitzschia (Bacillariophyceae)[END_REF][START_REF] Ishii | Growth characterization of non-photosynthetic diatoms, Nitzschia spp., inhabiting estuarine mangrove forests of Ishigaki Island, Japan[END_REF][START_REF] Karsten | Osmoprotectors in some species of Japanese mangrove macroalgae[END_REF]. Endopeptidases and aromatic compound degradation may facilitate the utilization of their complex carbon compounds.

Furthermore, transcriptional dynamics of the predicted secretome over a diel cycle (Fig. 2) revealed the presence of four different clusters. Genes in cluster 1 were transcribed at the beginning of the first light phase and genes in cluster 2 at the end of the dark phase and into the second light phase (Fig. 4G). Genes of cluster 3 were most strongly expressed in the middle and end of the first light phase, whereas genes in cluster 4 were relatively weakly expressed throughout day and night. These results suggest that stimuli including light and/or nutrients play a role in the regulation of these genes, which might either be a relict from the photosynthetic ancestor or a response to diel cycles of organic substances in the aquatic system occupied by N. putrida. There is only weak evidence of lateral transfer of secretome genes in N. putrida (figs. S14 and S15) with five genes of potential lateral origin (figs. S14 and S15). Thus, the origin of most secretome proteins in N. putrida likely was derived vertically from homologs of a photosynthetic ancestor.

DISCUSSION

N. putrida experienced a series of genetic adaptations toward a heterotrophic lifestyle. This diatom species took a step backward in one of the major evolutionary transitions, from photoautotrophs to heterotrophs, potentially relaxing selection on some of the now redundant gene networks and their functions. As expected, more than 50% of nuclear encoded plastid proteins have been lost in the N. putrida plastid proteome in comparison to its photosynthetic counterparts [START_REF] Gruber | Plastid proteome prediction for diatoms and other algae with secondary plastids of the red lineage[END_REF]. However, the total number of genes (~15,000) fell within the range of photosynthetic microalgae, and we found no evidence of pseudogene formation, genome streamlining [e.g., [START_REF] Wolf | Genome reduction as the dominant mode of evolution[END_REF]], gene family contraction (cf. birth-and-death hypothesis) [START_REF] Nei | Molecular Evolution and Phylogenetics[END_REF], or reductive genome evolution (Black Queen hypothesis) [START_REF] Morris | The Black Queen hypothesis: Evolution of dependencies through adaptive gene loss[END_REF]. The relatively large genome size is not unexpected given that N. putrida is a free-living osmotroph. This free-living lifestyle in a complex and highly variable coastal marine environment likely is the reason why a substantial number of genes including some photoreceptors, cell cycle regulators, and common plastid metabolic pathways usually present in photosynthetic diatoms have remained. Although some of the latter genes were still expressed, N. putrida appears to lack a diel growth cycle, which suggests that these cell-cycle regulators have neo/subfunctionalized. However, as a certain number of genes still appear to be regulated by light, osmotrophy potentially benefits from diel fluctuations of resources such as dissolved organic carbon in aquatic environments [START_REF] Ottesen | Multispecies diel transcriptional oscillations in open ocean heterotrophic bacterial assemblages[END_REF][START_REF] Aylward | Microbial community transcriptional networks are conserved in three domains at ocean basin scales[END_REF][START_REF] Frischkorn | Coordinated gene expression between Trichodesmium and its microbiome over day-night cycles in the North Pacific Subtropical Gyre[END_REF]. For photoautotrophs, it is important to regulate the cell cycle in accordance with diel cycles for optimizing photosynthesis and therefore cell proliferation (29-31, 52, 53). Without being reliant on light as its primary energy source, the osmotroph N. putrida no longer requires coordinating its cell cycle with diel cycles. Thus, after the loss of photosynthesis, the strict light-dependent regulation of gene expression might have become less important and gene expression therefore may have become predominantly regulated by other stimuli. Many photoreceptors are missing, but duplication of genes for bZIP transcription factors with PAS domains and genes for signal transduction and cellular regulatory roles such as adenyl/guanyl cyclase and cyclic nucleotide esterase domains was enriched in the N. putrida genome. Furthermore, the peroxisome-plastid interaction is no longer required after the loss of photosynthesis, giving rise to loss of carbon fixation in the context of glycolate recycling. In contrast, the ornithine-urea cycle likely remains to be functional to facilitate nitrogen recycling.

Gene family expansions and neo/subfunctionalizations appear to have played a prominent role in the adaptation to its different lifestyle given that many proteins predicted to be secreted have diversified in N. putrida, possibly to facilitate osmotrophy. Together, the marked change of lifestyle associated with the "devolution" did not result in reductive genome evolution as known from nonphotosynthetic plastid-bearing parasites.

METHODS

Genome assembly and construction of gene models

PacBio reads were assembled into contigs using Falcon (version 0.7.0) [START_REF] Chin | Phased diploid genome assembly with single-molecule real-time sequencing[END_REF] with a length cutoff of 7000 bp for seed reads and an estimated genome size of 33 Mbp. Genome size estimation was performed on the GenomeScope web server (http://qb.cshl.edu/genomescope/) based on the k-mer frequency distribution of Illumina reads calculated by JellyFish version 2.2.6 with a k-mer size of 21. The resultant primary and associate contigs were then subjected to Falcon_unzip Downloaded from https://www.science.org at Universite de Liege on May 04, 2022 (version 0.5.0) [START_REF] Chin | Phased diploid genome assembly with single-molecule real-time sequencing[END_REF], generating partially haplotype-phased contigs (primary contigs) and fully phased contigs (haplotigs). The assembly was polished using PacBio reads and Quiver program, followed by single-nucleotide polymorphism (SNP) and short insertion-deletion (indel) error correction using Pilon (version 1.2.2) with Illumina reads mapped by the Burrows-Wheeler Aligner (version 0.7.15) [START_REF] Walker | Pilon: An integrated tool for comprehensive microbial variant detection and genome assembly improvement[END_REF]. Indel errors in the vicinity of hetero-SNPs were further fixed manually, as they were difficult to be automatically corrected. Contigs derived from plastid and mitochondrial genomes were identified using BLASTN and separated from contigs derived from the nuclear genome.

RNA sequencing reads were trimmed under the parameters of ILLUMINACLIP:TruSeq3-PE.fa:2:30:10, LEADING:20 TRAILING:20, SLIDINGWINDOW:4:15, and MINLEN:75 using Trimmomatic (version 0.36) [START_REF] Bolger | Trimmomatic: A flexible trimmer for Illumina sequence data[END_REF]. The trimmed reads aligned to the assembled contigs using HISAT2 (version 2.0.4) [START_REF] Kim | HISAT: A fast spliced aligner with low memory requirements[END_REF]. They were provided to BRAKER2 gene annotation pipeline (version 2.0.3) [START_REF] Hoff | BRAKER1: Unsupervised RNA-Seq-based genome annotation with GeneMark-ET and AUGUSTUS[END_REF] as training data to be used for ab initio prediction of protein-coding genes. In addition, PASA (version 2.3.3) (56) was used to generate transcriptbased gene models by integrating de novo transcriptome assembly and genome-guided assembly using Trinity (version 2.5.0) [START_REF] Grabherr | Full-length transcriptome assembly from RNA-Seq data without a reference genome[END_REF]. The genome-guided assembly used the mapping result from HISAT2 with --dta option. TransDecoder (version 5.0.2) (58) was used to extract protein coding regions from PASA result with the alignment files from BlastP (version 2.7.1) (59) against UniRef90 with -evalue 1e-5 option and hmmscan (http://hmmer.org/, version 3.1b2) against Pfam (60) database.

The gene models that overlapped with the results from BRAKER were removed using BlastP with evalue 1e-5 option, and the remaining gene models were merged with the BRAKER gene models to generate the final gene annotation. Transposable elements in the NIES-4239 genome were searched by RepeatMasker (version 4.9.0) using Dfam3.1 and RepBase-20170127 as reference repeat libraries [START_REF] Tarailo-Graovac | Using RepeatMasker to identify repetitive elements in genomic sequences[END_REF]. The predicted gene set was available in Dryad (https://doi. org/10.5061/dryad.j3tx95xft).

The integrity of gene annotation was assessed by BUSCO (version 3.0.2) [START_REF] Waterhouse | BUSCO applications from quality assessments to gene prediction and phylogenomics[END_REF] and the Eukaryota odb9 (version 2) dataset. The manipulation of SAM/BAM file was used by SAMtools (version 1.9). The sequence files of gene region from gff file were used by GffRead (version 0.9.11) [START_REF] Pertea | GFF utilities: GffRead and GffCompare[END_REF].

Organellar genome annotation was performed by comparison with previously sequenced organellar genomes of nonphotosynthetic diatoms [START_REF] Kamikawa | Proposal of a twin arginine translocator system-mediated constraint against loss of ATP synthase genes from nonphotosynthetic plastid genomes[END_REF]. Gene sets and their arrangements of the plastid and mitochondrial genomes sequenced in this study were found to be identical to previously sequenced nonphotosynthetic diatoms [START_REF] Kamikawa | Proposal of a twin arginine translocator system-mediated constraint against loss of ATP synthase genes from nonphotosynthetic plastid genomes[END_REF]. Assembled genomes were deposited to DNA Data Bank of Japan (http://getentry.ddbj.nig.ac.jp/) under the accession numbers BLYE01000001 to BLYE01000234 for the nuclear genome, LC600866 for the mitochondrial genome, and LC600867 for the plastid genome.

Functional annotation

The predicted protein coding genes were annotated using InterProScan, and RPS-BLAST search was performed against KEGG orthology database [START_REF] Jones | InterProScan 5: Genome-scale protein function classification[END_REF][START_REF] Marchler-Bauer | CD-Search: Protein domain annotations on the fly[END_REF]. KO identifiers for Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways were assigned using KEGG Automatic Annotation Server [START_REF] Moriya | KAAS: An automatic genome annotation and pathway reconstruction server[END_REF]. Transporter proteins were annotated with TransportTP (66) followed by manual curation. Reference proteome datasets for three photosynthetic diatom species were obtained from the JGI Genome Portal: P. tricornutum CCAP 1055/1 v2.0 (Phatr2_bd_unmapped_GeneModels_FilteredModels1_ aa.fasta and Phatr2_chromosomes_geneModels_FilteredModels2_ aa.fasta, 10,402 protein sequences in total), T. pseudonana CCMP 1335 (Thaps3_bd_unmapped_GeneModels_FilteredModels1_aa.fasta and Thaps3_chromosomes_geneModels_FilteredModels2_aa.fasta, 11,776 sequences), and F. cylindrus CCMP 1102 (Fracy1_GeneModels_ FilteredModels3_aa.fasta, 21,066 sequences). KEGG and KOG annotation was performed with them in the same manner as NIES-4239. Other details for annotation of CAZyme, cyclins, cyclin-dependent kinases, bZIP transcription factors, photoreceptor proteins, mitochondrial proteins, plastid proteins, and secretome proteins are described in the Supplementary Materials. Evolutionary analyses, comparative transcriptome analyses under the 12-hour light and 12-hour dark condition, those in different carbon sources, and biochemical experiments for lipids, fatty acids, and quinones are also described in the Supplementary Materials. Transcriptome data obtained in this study were deposited to DNA Data Bank of Japan (https://ddbj.nig.ac.jp/resource/bioproject/PRJDB11016 and https://ddbj.nig.ac.jp/resource/bioproject/PRJDB12553).

Fig. 1 .

 1 Fig. 1. The heterotrophic diatom N. putrida and its plastid proteome. (A) The frustule view of N. putrida. Bar, 10 m. (B) Estimated plastid proteome size in three diatoms. Light and dark gray bars show low and high confident plastid-targeted proteins identified by ASAFind[START_REF] Gruber | Plastid proteome prediction for diatoms and other algae with secondary plastids of the red lineage[END_REF], respectively. Data of two photosynthetic diatoms P. tricornutum and T. pseudonana are derived from the previous study[START_REF] Gruber | Plastid proteome prediction for diatoms and other algae with secondary plastids of the red lineage[END_REF]. (C) Unique and shared plastid-targeted orthogroups. Highlighted in red is the orthogroup exclusively shared by the two photosynthetic diatoms. (D) Predicted metabolic map of the nonphotosynthetic plastid. Representative pathways found in photosynthetic diatom species are shown. Green and light gray arrows show the presence and absence of the responsible protein sequences for the reactions in the genome, respectively. Amino acids are highlighted in red. Abbreviations are described in the Supplementary Materials.

  Photorecepter

Fig. 2 .

 2 Fig. 2. Loss of genes for the plastid-peroxisome metabolic flow and photoreceptors. (A) Metabolic interactions between a mitochondrion and a nonphotosynthetic plastid and between a mitochondrion and a peroxisome. Black, orange, and blue arrows show presence of responsible protein sequences for the reactions in a plastid, a mitochondrion, and a peroxisome, respectively, while light gray arrows show absence of responsible protein sequences. Dashed arrows show possible interorganellar metabolic flows. Abbreviations are described in the Supplementary Materials. (B) Photoreceptor and cell-cycle genes in the N. putrida genome. The other genes are shown in fig. S5. Light green and light gray boxes show the presence and absence of corresponding genes, respectively. (C) Growth of the heterotrophic diatom under the different light conditions. Closed boxes show growth in the continuous dark condition, while open boxes show growth in the light-dark condition. Shaded in gray are the dark periods in the light-dark cultivation conditions. (D) Left: Heatmap showing the reproducible expression patterns of genes (Pearson's correlation coefficient < 0.9). k-means clustering was calculated for each gene based on reads per kilobase of transcript per million mapped reads (RPKM) + 1 values, which were transformed to log 2 and centered by median values. Yellow and blue indicate up-regulation and down-regulation of the gene, respectively. Right: The line graphs showing expression pattern of genes in each cluster. The colored line indicates the average value of the expression patterns. LogFC, log fold change.

  Silicon transporter (Sit) family P-type ATPase (P-ATPase) superfamily Voltage-gated ion channel (VIC) superfamily Amino acid/auxin permease (AAAP) family Drug/metabolite transporter (DMT) superfamily Mitochondrial carrier (MC) family ATP-binding cassette (ABC) superfamily Major facilitator superfamily (MFS)

Fig. 3 .

 3 Fig. 3. Diversity of transporters and carbohydrate active enzymes in N. putrida. (A) Distribution of the number of transporters in each transporter family of diatoms. Differences in the distributions among species were tested by the Wilcoxon signed-rank test corrected with the Benjamini-Hochberg procedure (P < 0.05), but there is no significant difference. Outliers were omitted in the boxplot. Nonphotosynthetic species are highlighted in gray. (B) Distribution of the number of transporters in each transporter family of Alveolata. Details are described in (A). (C) Distribution of the number of transporters in each transporter family of green algae (Trebouxiophyceae). Details are described in (A). (D) The gene number of transporters in the 12 most abundant transporter families of N. putrida. (E) Silicon transporter (SIT) genes tandemly located in the contig 000000F. SIT genes are highlighted in light red with the gene IDs. (F) Glycoside hydrolase (GH) families from the Carbohydrate Active enZyme (CAZy) database focused on diatoms. The diagram shows a heatmap of CAZyme prevalence in each taxon (number of a particular CAZyme family divided by the total number of CAZyme genes in the organism); the white to blue color scheme indicates low to high prevalence, respectively. Dendrograms (left and top) show respectively the relative taxa proximity with respect co-occurrence of CAZyme families and the co-occurrence of CAZyme families with one another within genomes. (G) GH114 genes tandemly located in the contig 000022F. GH114 genes are highlighted in light green with the gene IDs.

Fig. 4 .

 4 Fig. 4. Secretome of nonphotosynthetic algae. (A)The number of secretome tribes of diatoms, including at least four sequences, clustered by TribeMCL[START_REF] Karsten | Osmoprotectors in some species of Japanese mangrove macroalgae[END_REF]. Different colors represent tribe categories as follows: 1, species specific tribes; 2 to 4, tribes shared by two to four species, respectively. OTUs, operational taxonomic unit. (B) Proportion of each tribe category in diatoms. Details are described in (A). (C) Distribution of the number of protein sequences in each secretome tribe in diatoms. Outliers were omitted in the boxplot. The Wilcoxon signed-rank test corrected with the Benjamini-Hochberg procedure was used for tests of statistical significance. (D) The number of secretome tribes in green algae (trebouxiophytes), including at least four sequences, clustered by TribeMCL[START_REF] Karsten | Osmoprotectors in some species of Japanese mangrove macroalgae[END_REF]. Different colors represent tribe categories as follows: 1, species specific tribes; 2 to 5, tribes shared by two to five species, respectively. (E) Proportion of each tribe category in green algae. Details are described in (D). (F) Distribution of the number of protein sequences in each secretome tribe in green algae. Details are described in (C). (G) Expression of the 10 largest tribes in N. putrida during the 25 hours of cultivation. Genes in the tribes could be divided into four clusters. Details are described in Fig.2D.

  Cultivation, DNA and RNA extraction, and sequencing N. putrida NIES-4239 was cultivated in the Daigo's IMK medium (Wako) including 1% Luria-Bertani medium based on the artificial seawater made with MARINE ART SF-1 (Osaka Yakken Co.) at 20°C under the 12-hour light and 12-hour dark conditions: 50 mol photons/m 2 per second with plant cultivation light-emitting diode light (BC-BML3, Biomedical Science). DNA was extracted with the Extrap Soil DNA Kit Plus version 2 (Nippon Steel). Total DNA was subjected to library construction with TruSeq DNA PCR-Free (350; Illumina) and to 151-bp paired-end sequencing by HiSeqX, resulting in 660 million paired-end reads, and to PacBio RSII, with SMRT cell 8Pac v3 and DNA Polymerase Binding Kit P6 v2, in Macrogen, resulting in 1.3-Gb subreads. Total RNA was extracted with TRIzol (Sigma-Aldrich) according to the manufacturer's instruction and was subjected to library construction with TruSeq RNA Sample Prep Kit v2 (Illumina) and 101-bp paired-end sequencing by HiSeq 2500, resulting in 107.5 million paired-end reads.

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than in the photosynthetic diatom genomes (188 in N. putrida, 142 in F. cylindrus, 118 in P. tricornutum, and

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Acknowledgments: We thank F. Hayashi for the manual correction of the genome assembly and the gene annotation for the secretome. Computational work was, in part, performed at the SuperComputer System, Institute for Chemical Research, Kyoto University, and the NIG supercomputer at the National Institute of Genetics. Funding: This work was supported by JSPS Grants-in-Aid for Scientific Research (B) (19H03274 to R.K., 20H03305 to T.N., and 17H03723 to G.T.); JSPS Grants-in-Aid for Early-Career Scientists (20K15783 to Y.T.); JSPS Grant-in-Aid for Challenging Research (Exploratory) (21K19303 to G.T.); JSPS Grants-in-Aid for Scientific Research (A) (18H03743 to Y.K.); JSPS Grants-in-Aid for Scientific Research on Innovative Areas, Platform for Advanced Genome Science (16H06279 to R.K., T.Moch, M.S., Y.T., and Y.N.); NERC (NE/R000883/1 to T. Mock, C.v.O., A.T., K.S., and S.S.); School of Environmental Sciences at the University of East Anglia, UK (to T. Mock, C.v.O., A.T., K.S., and S.S.); The Gatsby Charitable Foundation and Biotechnology and Biological Sciences Research Council (BBSRC) (to S.K. and J.W.); and NIG-JOINT (7A2017, 6A2018, and 30A2019 to R.K.). Author contributions: Conceptualization: R.K., G.T., T. Mock, and Y.N. Methodology: R.K., T. Mock, and Y.N. Investigation: R.K., Y.T., T.Moch., M.S., G.T., Y.N., U.C., B.H., V.L., D.M., S.S., K.S., A.T., C.v.O., T. Mock, K.O., K.-i.I., M.Kay., T.A., Y.K., and J.W. Visualization: R.K., R.O., K.K., T.N., U.C., S.S., K.S., A.T., and C.v.O. Supervision: S.-y.M., M.Kat., H.M., S.M., and S.K. Writing-original draft: R.K., C.v.O., and T. Mock. Writingreview and editing: All the authors. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper are available in DDBJ and Dryad as described in Methods. Submitted 15 March 2021 Accepted 16 March 2022 Published 29 April 2022 10.1126/sciadv.abi5075 Downloaded from https://www.science.org at Universite de Liege on May 04, 2022

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Genome evolution of a nonparasitic secondary heterotroph, the diatom Nitzschia putrida


Nhat Tam Vo

Christian Herrero

Régis Guillot

Tanya Inceoglu

Winfried Leibl

Martin Clémancey

Patrick Dubourdeaux

Geneviève Blondin

Ally Aukauloo

Marie Sircoglou

Intercepting a Transient Pyridine N-Oxide Fe(III) Species in Catalytic Oxygen Atom Transfer Reaction

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

In the context of bioinspired OAT catalysis, we developed a tetradentate dipyrrinpyridine ligand, halfway between hemic and non-hemic models. The catalytic activity of the iron(III) derivative was investigated in presence of iodosylbenzene. Unexpectedly, MS, EPR, Mössbauer, UV-Visible and FTIR spectroscopic signatures supported by DFT calculation provide convincing evidence for the reversible formation of a relevant Fe III -O-NPy intermediate.

Oxygen Atom Transfer (OAT) reactions are important in chemical synthesis and biology where high valent metal-oxos are key catalytic species. 1 Bioinspired models have been investigated to replicate the reactivity and selectivity of hemic and non-hemic enzymes. After much effort, chemists have unravelled facets of the structure and function relationship of the catalytic intermediates that affect substrate oxidation. 2 While the reactivity studies have long been focused on +IV and +V oxidation state metal-oxo intermediates, multiple active oxidant mechanisms have also been proposed (Scheme 1). 1a,3 For instance, iron(III) iodosylarene adducts have been considered as alternate active species. 4 In-depth studies, implying chemical variation of the iodosylarene, 5 X-ray structure characterization of such intermediate 6 and DFT calculations 7 have contributed to support this pathway. In parallel, pyridine N-oxides have been used as terminal oxidant in molecular metal complexes assisted OAT reactions. 8 Che and coll. have in particular reported interesting OAT activities of a wide variety of organic substrates by combining orthosubstituted pyridine N-oxide with Ru catalysts. 9 Fukuzumi and coll. also demonstrated the photoactivation of an embarked outer sphere pyridine N-oxide fragment in a ruthenium-based complex to transfer the oxygen atom to an organic substrate. 10 More generally, the propensity to use heteroarene N-oxides as oxygen source in organic reactions was recently highlighted by Pospech. In this review, no mention is made of inner-sphere bound pyridine N-oxide as potential catalytic species towards OAT reactions. 11 Yet, in several investigations, iron-N-oxide species have been characterized. McKenzie and coll. reported the chemical alteration of an Fe(V)-oxo species in the form a metal bonded tertiary amine N-oxide that was characterized crystallographically. 12 In another example, the flexibility of a hexadentate ligand was proposed to favour the stabilization of an electrophilic oxyl group by a pendant pyridyl arm. 6 More recently the lack of reactivity of a putative [LFeO] 2+ intermediate was attributed to the formation of pyridine Noxide iron(III) complex, devoid of any catalytic activity. 13 Henceforth, in the actual state of the art, there is no report of any case where post synthetic iron N-oxide species can themselves be involved in a catalytic OAT reaction to organic substrates. On the contrary, their formation is rather considered as deleterious pathway. In this study, we report the synthesis of an iron(III) complex supported by a semi-hemic dipyrrinpyridine ligand, shorthanded as DPPy under its deprotonated form. The design of the ligand was directed to exclude alkyl chains in the backbone to limit its flexibility and increase its robustness towards self-oxidation. 14 We found that in presence of iodosylbenzene, [Fe III DPPyCl2] could promote the oxidation of a fair variety of substrates. In the process to unravel the nature of the putative oxidizing reactive species, no spectroscopic signatures for a highly oxidized Fe-oxo intermediate were detected. However, MS and IR collected data provide concurring evidence for the chemical formulation of an The DPPyH ligand and the [Fe III DPPyCl2] complex were prepared by adapting reported procedures (see ESI). Dark blue single crystals of the complex were obtained from an Et2O/ACN mixture. The molecular structure was determined by X-ray diffraction (XRD) analysis (Fig. 1). The coordination sphere around the iron ion is a distorted octahedron with four nitrogen atoms in a plane and two pseudo-axial chloride ligands held at an angle of 152° from the metal. The open structure of the DPPy ligand leads to a trapezoidal N4 coordinating scheme supported by two short metal-ligand bond with the dipyrrin pointing at Fe-N1 and Fe-N2 = 2.048(3) Å and two longer ones involving the pyridine rings (Fe-N3 and Fe-N4 = 2.238(4) Å). These bond lengths are respectively equivalent to those of Fe(III) porphyrin complexes (av. 2.02 Å) and longer than those encountered classically in pyridine containing ligands (av. 2.15 Å, see ESI). The XRD analysis of crystals grown in acetone indicated two different conformations, one where the DPPy backbone is severely twisted by 14.70° while the second exhibits a dissymmetric coordination pattern (See ESI and Fig. S1-S4). These structural snapshots point to a certain displacement of the iron(III) to adapt within the trapezoidal coordinating N4 pattern supported by the DPPy ligand. The consequence of this, results in the distancing of the iron ion away from one of the pyridine group as schematized in Fig. 1. The X-band EPR spectrum of the complex was recorded at 10 K in acetonitrile and revealed three signals with g values at 9.1, 5.1 and 3.7 consistent with high spin (S = 5/2) Fe(III) species (Fig. S6). This spectrum was best simulated with the contribution of two high spin species probably originating from subtle structural difference as observed in the solid state. The catalytic activity of [FeDPPyCl2] was evaluated in the presence of iodosylbenzene as an oxygen atom donor. Under unoptimized conditions, quantitative oxidation of triphenylphosphine and thioanisole were observed at room temperature. More challenging substrates such as cyclooctene, toluene or cyclohexane, were also oxidized with good to modest yields (Scheme 2, Table S3). The observed reactivity matches those reported for hemic and non-hemic OAT catalysts, prompting us to further investigate the nature of the catalytic species involved in this unique hybrid hemic/non-hemic system. Our first experiment consisted in the monitoring of the electronic absorption spectral features of a solution of complex [FeDPPyCl2] in acetonitrile upon addition of a stoichiometric amount of PhIO at room temperature. The starting iron(III) complex presents two strong absorption bands in the region spanning from 500 to 700 nm. Such features are also present for the free ligand albeit with different amplitudes and energies. 15 Accordingly, the absorption bands for the [FeDPPyCl2] complex are likely attributed to metal-ligand and/or ligand-ligand charge transfer bands. As depicted in Fig. 2, addition of PhIO caused the instant bleach of the precursor band at 650 nm and apparition of a strong absorption band at 636 nm (e » 20 000 L mol -1 cm -1 ) with a concomitant drop in intensity of the initial absorption band at 589 nm (e = 21 600 L mol -1 cm -1 ). Coincidentally, weaker absorption bands have been reported for non-hemic iron-oxo intermediates in this spectral window although with lower e values to confound with the actual bands. 16 This new prominent spectral feature then faded away within 15 mins to rest the initial UV-vis spectrum of [FeDPPyCl2] (lmax = 589 nm, e = 21 600 L mol -1 cm -1 ) albeit with slightly different relative intensities. Such an evolution can be related to the generation of an active species that would react with the solvent to regenerate the starting complex. During this process, some modification of the coordination sphere of iron may occur, explaining the slight difference observed in the recovered absorption spectrum. In another experiment, when triphenylphosphine was added to the oxidized species, the decay was accelerated by a factor of 5 (3 mins) while triphenylphosphine oxide was detected by Mass spectroscopy (MS) and NMR. This result put us on track that the monitored intermediate, denoted as species I is a relevant OAT agent. We then interrogated the oxidation state of this intermediate using X-band EPR spectroscopy. As can be seen in Fig. 2, the initial signal of the precursor [FeDPPyCl2] complex was replaced by a broad rhombic signal at g = 4.28 (E/D = 0.33) with an estimated conversion of around 60%. In addition, no signal was detected in parallel mode most probably precluding an integer spin state for the reactive intermediate. Besides no Fe-oxo intermediate could be detected even upon reaction at lower temperature. Unequivocally, addition of triphenylphosphine conducted to the disappearance of the signal at 4.28 with the concurrent recovery of the initial EPR signals of the precursor. These results therefore suggest that the reactive species I contains a high spin Fe(III) ion in a rhombic environment. Further confirmation for the oxidation state of I comes from the Please do not adjust margins

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Mössbauer analysis of a 57 Fe enriched intermediate I at 6K (Fig. S8). Upon addition of PhIO, the broad dissymmetric doublet , (d = 0.45 mm s -1 , DEQ = 0.78 mm s -1 ) of the starting complex gives rise to a new spectrum that can be described as the main contribution of a broad sextet extending from -8 to 9 mm s -1 (64%, d = 0.46 mm s -1 , DEQ = 0.61 mm s -1 ) attributed to the signature of a S=5/2 species and a quadrupolar doublet (39%, d = 0.51 mm s -1 , DEQ = 1.59 mm s -1 ) most probably arising from a magnetically silent µ-oxo dimeric species generated under the high concentration of the Mössbauer analysis. 17 No genuine Fe(V) or Fe(IV)=O signatures were identified therefore ruling out such formulation for the transient species previously detected by UV and EPR. Interestingly, upon ageing of the previous solution, the main magnetic Mössbauer signal disappeared to give a 1:1 mixture of the starting signal and the diamagnetic doublet (Fig. S8 bottom). These results again provide support for the formation of an Fe(III) transient species, I. At this juncture, we cogitated on a first alternative for the description of an iron(III) species that can behave as an oxygenated atom source. For this, we turned our attention to the formation of a Fe(III) iodosylbenzene adduct that has already been reported in literature by Nam and McKenzie. Mass spectrometry analysis is extremely sensitive and can permit the detection of unexpected highly active intermediates or chemically altered forms of the catalyst upon reaction with the chemical oxidant for instance. A solution of [FeDPPyCl2] in acetonitrile was treated with PhIO and followed by High Resolution ESI-MS. A peak of weak intensity was detected at 773.9389 that corresponds to the iodosylbenzene adduct. However, several more prominent peaks were also present.

Although the relative intensities of the mass-to-charge ratio do not necessarily reflect the actual concentration of the species in solution, we were piqued to investigate the nature of these signals. First, we identified a peak pointing at 534.0194 that matches the formula [FeDPPy(O)-H] + and can tentatively be assigned to a C-H hydroxylation product (see ESI). Such hydroxylation of aromatic rings has been observed in prior studies 18 and might partially account for the incomplete recovery of the initial absorption spectrum described above. Two peaks of interest were also evidenced at 569.9954 and 580.0242 that corresponds to the chemical formulations [FeDPPy(O)Cl] + and [FeDPPy(O)(HCOO)] + respectively (Fig. S9). These peaks were sensitive when labelled H2 18 O was added after reaction with PhIO. This result supports the fact that the transferred oxygen atom is chemically labile towards exchange within these species (Fig. S10). Having previously ruled out the formation of iron oxo species, an additional signal peaking at 503.0879 caught our attention. This peak relates to a molecular fragment [(DPPyH(O)+Na] + , i.e. the sodium adduct of the free ligand with an inserted oxygen atom. 19 With this formulation, a reasonable site for such a reaction is one of the pyridine groups to form an N-O pyridine noted as (DPPyO). Such oxygen insertion into a metal-heteroatom bond have been well documented with pyridine-amines containing ligands. 20 In our case, the rigid conjugated acyclic skeleton of the DPPy ligand resulting in a loose coordination of the iron(III) ion is probably the crucial structural factor that facilitates the oxygen insertion to form a [Fe III (DPPyO)Cl2] species. Similar oxygen atom insertion was attested crystallographically by Lau and coll. with an analogous quarterpyridine ligand to form a quaterpyridinedioxide ruthenium complex acting as the active species for the water oxidation reaction. 20b In our case, all attempts to crystallize such an intermediate failed due to its short life time and its high solubility. However, infrared spectroscopy provided supportive evidence for the presence of the N-O bound fragment. Compared to the initial [FeDPPyCl2] complex, two new bands sensitive to 18 O labelling were clearly apparent at 1226 and 845 cm -1 on the IR spectrum of a [FeDPPyCl2] / PhIO mixture (Fig. 3 andS12). These two signals can be respectively assigned to the stretching and bending vibration mode of the pyridine N-OFe bond by comparison to the values reported for Fe(OPy)6(ClO4)3 (1214, 839 cm -1 ). 21 Importantly, these bands were absent in aged solutions and can therefore be related to a transient species.

With a systematic approach to analyze the reactivity pattern with other terminal oxidants, we assessed the reactivity with mCPBA and NaClO. In the presence of mCPBA, a rapid degradation of the precursor was observed. However, when NaClO was used instead of PhIO, very similar spectra were obtained by UV-Vis, EPR, MS and IR spectroscopy (Fig. S13-S16).

As with PhIO, the new spectral features were bleached upon addition of triphenylphosphine (Fig. S14). Please do not adjust margins Please do not adjust margins frequencies were isotopically shifted to higher energies (Fig. 3, S18-19), thus comforting our hypothesis. Although a full study of the mechanistic pathway leading to the formation of the pyridine N-oxide adduct is beyond the scope of this paper, we theoretically investigated the putative iron(V)-oxo and iron(III)iodosylbenzene intermediates (Fig. S21). By contrast, these notional structures were respectively found 35 and 21 kcal mol -1 less stable than the computed [Fe III (DPPyO)Cl2], indicating that such species if they are formed, should alter rapidly. Of particular relevance, we noticed the departure of one pyridine arm in the Fe-oxo adduct. As stated above, the DPPy ligand probably provides a somewhat rigid N4 coordinating cavity and therefore cannot flex enough to accommodate the smaller highly oxidized iron(IV) or iron(V) ion. Such a structural feature in the designed DPPy ligand agrees with a possible intramolecular OAT to the free pyridine.

In this study, we have investigated the OAT reactivity of a newly developed iron(III)-dipyrrin-dipyridine complex, [Fe III DPPyCl2]. UV-vis, Mössbauer and EPR spectroscopies point to the formation of an Fe(III) active species, while ESI-MS and IR and analysis corroborated by DFT helped us identifying the oxygenation of a Fe-Npy bond. Oxygen insertion in ligand-Fe bonds has only been observed in rare cases and were most often considered as dead ends devoid of any reactivity. 13,22 Herein, upon treatment of [Fe III DPPyCl2] with an oxygencontaining oxidant such as PhIO, we propose the formation of intramolecular Fe III -O-NPy intermediate that still allows Odelivery to various organic substrates. Such a behavior has to our knowledge only been observed in the case of Ru complexes and under light irradiation. 10 Whether the mechanism involving the [Fe III (DPPyO)Cl2] intermediate I for the O-atom delivery to organic substrates proceeds via the classic generation of a potent Fe=O or a concerted pathway still remain to be elucidated. Future modifications of the DPPy backbone will help us to decipher and optimize the unusual oxidation process reported herein. 23 Work along these lines is in progress.

Fig. 2

 2 Fig.2Spectral changes of a [FeDPPyCl2] solution in ACN before (blue) and after (green) oxidation by PhIO, followed by its degradation (red) or reaction with PPh3 (black). Left: UV-vis, Inset: degradation kinetics; Right: X-band EPR at T=10K.

Fig. 1

 1 Fig. 1 Left: ORTEP view of the XRD structure of [FeDPPyCl2], single crystals grown in Et2O/ACN, H omitted, ellipsoids at 30%; Right: Scheme showing Fe displacement in the N4 cavity upon changing the solvent of crystallization to acetone.

  Fig. 3 FTIR spectra of [FeDPPyCl2] before (black) and after (green) addition of PhIO; Inset: computed [Fe(DPPyO)Cl2] structure, Table: Experimental isotope sensitive bands compared to the calculated vibration modes involving N-O bonds

  These intrigues are elucidated in what follows with the support of DFT calculations.

	transient species.
	a. ICMMO, Université Paris Saclay, CNRS, F-91405 Orsay Cedex, France.
	b. Institute for integrative Biology of the Cell (I2BC), CEA, CNRS Université Paris-
	Saclay, UMR 9198, F-91191, Gif-sur-Yvette, France.
	c. Laboratoire de Chimie et Biologie des Métaux, Univ. Grenoble Alpes, CNRS, CEA, IRIG, 17 rue des Martyrs, F-38000 Grenoble, France. Electronic Supplementary Information (ESI) available. See DOI: 10.1039/x0xx00000x	[Fe III (DPPyO)Cl2] spectroscopy also nails the formation of a high spin Fe(III) intermediate. EPR and Mössbauer

  Main oxidation products obtained upon catalytic oxidation by[FeDPPyCl2] with PhIO (20 eq.) in acetonitrile. Conversions determined by GC.

	PPh 3 100% (1 min) O	O	O	OH	OH	OH
	O
	S
	95% (2h)	46% (24h)	35% (24h)	93% (24h)	15% (24h)	3% (24h)

Ph

Scheme 2.

We thank LabEx CHARM 3 AT & ARCANE, HPC resources (GENCI-A0070810977), DataCenter@UPSud. GB thanks Dr V.Maurel for EPR recordings.

Notes and references