Bram Danneels
email: bram.danneels@inrae.fr

Monique Blignaut

Guillaume Marti

Simon Sieber

Peter Vandamme

Marion Meyer

Aurélien Carlier
email: aurelien.carlier@inrae.fr

Danneels

Bram

Cyclitol metabolism is a central feature of Burkholderia leaf symbionts

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

Interactions with microbes play an important part in the evolution and ecological success of plants. For example, mycorrhizal associations are present in a vast majority of land plants, and the association with nitrogen-fixing bacteria provided legumes with an important evolutionary advantage [START_REF] Brundrett | Mycorrhizas in Natural Ecosystems[END_REF][START_REF] Van Rhijn | The Rhizobium-plant symbiosis[END_REF][START_REF] Vessey | Root-based N2-fixing Symbioses: Legumes, Actinorhizal Plants, Parasponia sp. and Cycads[END_REF][START_REF] Smith | The symbionts forming arbuscular mycorrhizas[END_REF]. Nevertheless, microbes may also be harmful for plants as microbial pathogen interactions are responsible for major crop losses [START_REF] Dangl | Plant pathogens and integrated defence responses to infection[END_REF][START_REF] Mccann | Skirmish or war: the emergence of agricultural plant pathogens[END_REF]. Many plant-microbe interactions only occur temporarily: contacts between microbes and the host are often limited to a sub-population or a specific developmental phase of the host. However, in some associations microbes are transferred from parents to offspring in a process called vertical transmission, resulting in permanent associations with high potential for co-evolution [START_REF] Gundel | Vertically transmitted symbionts as mechanisms of transgenerational effects[END_REF]. While verticallytransmitted microbes are common in the animal kingdom, they have been more rarely described in plants [START_REF] Fisher | The evolution of host-symbiont dependence[END_REF].

A particular case of vertically transmitted microbes in plants are the bacterial leaf endophytes found in three different plant families: the monocot Dioscoreaceae, and the dicot Rubiaceae and Primulaceae. In the genera Psychotria, Pavetta, Sericanthe (Rubiaceae) and Ardisia (Primulaceae) this association may manifest in the form of conspicuous leaf nodules that house extracellular symbiotic bacteria [START_REF] Miller | Bacterial Leaf Nodule Symbiosis[END_REF][START_REF] Van Oevelen | Identification of the bacterial endosymbionts in leaf galls of Psychotria (Rubiaceae, angiosperms) and proposal of "Candidatus Burkholderia kirkii" sp. nov[END_REF][START_REF] Lemaire | Identification, origin, and evolution of leaf nodulating symbionts of Sericanthe (Rubiaceae)[END_REF][START_REF] Lemaire | Identification of the bacterial endosymbionts in leaf nodules of Pavetta (Rubiaceae)[END_REF][START_REF] Ku | Phylogenetic and Cophylogenetic Analyses of the Leaf-Nodule Symbiosis in Ardisia Subgenus Crispardisia (Myrsinaceae): Evidence from Nuclear and Chloroplast Markers and Bacterial rrn Operons[END_REF]. In some of these systems, the symbiont was detected in seeds, indicating that they can be transmitted vertically [START_REF] Miller | Location and distribution of symbiotic bacteria during floral development in Ardisia crispa[END_REF][START_REF] Sinnesael | Detection of Burkholderia in the seeds of Psychotria punctata (Rubiaceae) -Microscopic evidence for vertical transmission in the leaf nodule symbiosis[END_REF]. Molecular analysis of the leaf nodules revealed that all endophytes are members of the Burkholderia sensu lato, more specifically to the newly defined Caballeronia genus [START_REF] Van Oevelen | Identification of the bacterial endosymbionts in leaf galls of Psychotria (Rubiaceae, angiosperms) and proposal of "Candidatus Burkholderia kirkii" sp. nov[END_REF][START_REF] Ku | Phylogenetic and Cophylogenetic Analyses of the Leaf-Nodule Symbiosis in Ardisia Subgenus Crispardisia (Myrsinaceae): Evidence from Nuclear and Chloroplast Markers and Bacterial rrn Operons[END_REF]. Similar leaf endophytes, also belonging to the Burkholderiaceae, are present in Rubiaceae species that do not form leaf nodules, including some Psychotria species [START_REF] Lemaire | Screening for leaf-associated endophytes in the genus Psychotria (Rubiaceae)[END_REF][START_REF] Verstraete | Symbiotic ß-Proteobacteria beyond Legumes: Burkholderia in Rubiaceae[END_REF]. To date, only one symbiont of Rubiaceae and Primulaceae has been cultivated: the endophyte of Fadogia homblei, which has been identified as Paraburkholderia caledonica [START_REF] Verstraete | Endophytic Bacteria in Toxic South African Plants: Identification, Phylogeny and Possible Involvement in Gousiekte[END_REF]. Interestingly, members of P. caledonica are also commonly isolated from the rhizosphere or soil and have been detected in leaves of some Vangueria species [START_REF] Verstraete | Intraspecific variation in Burkholderia caledonica: Europe vs. Africa and soil vs. endophytic isolates[END_REF]. Speculations about possible functions of these leaf symbioses have long remained unsubstantiated because efforts to isolate leaf nodule bacteria or to culture bacteria-free plants were unsuccessful [START_REF] Miller | Bacterial Leaf Nodule Symbiosis[END_REF]. Recently, sequencing and assembly of leaf symbiont genomes of several Psychotria, Pavetta or Ardisia species allowed new hypotheses about the ecological function of leaf symbiosis. Leaf symbiotic Candidatus Burkholderia crenata associated with Ardisia crenata, are responsible for the production of FR900359, a cyclic depsipeptide with potent bioactive and insecticidal properties [START_REF] Fujioka | Structure of FR900359, a cyclic depsipeptide from Ardisia crenata sims[END_REF][START_REF] Carlier | The genome analysis of Candidatus Burkholderia crenata reveals that secondary metabolism may be a key function of the Ardisia crenata leaf nodule symbiosis[END_REF]. Similarly, analysis of the genome of Ca. Burkholderia kirkii (Ca. B. kirkii), the leaf symbiont of Psychotria kirkii, revealed a prominent role of secondary metabolism [START_REF] Carlier | The eroded genome of a Psychotria leaf symbiont: hypotheses about lifestyle and interactions with its plant host[END_REF]. In this species, two biosynthetic gene clusters harboured on a plasmid encode two homologs of a 2-epi-5-epi-valiolone synthase (EEVS). EEVS are generally required for the production of cyclitol sugar analogs, a family of bioactive natural products with diverse targets [START_REF] Mahmud | The C7N aminocyclitol family of natural products[END_REF][START_REF] Mahmud | Progress in aminocyclitol biosynthesis[END_REF]. Ca. B. kirkii is likely involved in the synthesis of two cyclitol metabolites: kirkamide, a C7N aminocyclitol with insecticidal properties, and streptol glucoside, a derivative of valienol with broad allelopathic activities [START_REF] Sieber | Isolation and Total Synthesis of Kirkamide, an Aminocyclitol from an Obligate Leaf Nodule Symbiont[END_REF][START_REF] Georgiou | Leaf nodule endosymbiotic Burkholderia confer targeted allelopathy to their Psychotria hosts[END_REF]. Similarly, representative genomes of Candidatus Burkholderia humilis, Candidatus Burkholderia pumila, Candidatus Burkholderia verschuerenii, Candidatus Burkholderia brachyanthoides, Candidatus Burkholderia calva and Candidatus Burkholderia schumanniana associated with leaf nodules of various Psychotria and Pavetta species, encode putative EEVS gene clusters [START_REF] Pinto-Carbó | Evidence of horizontal gene transfer between obligate leaf nodule symbionts[END_REF]. The broad conservation of EEVS in otherwise small genomes suggests that C7 cyclitol compounds are important for leaf symbiosis in these species.

C7 cyclitols are a group of natural products derived from the pentose phosphate pathway intermediate sedoheptulose-7-phosphate (SH7P) [START_REF] Mahmud | The C7N aminocyclitol family of natural products[END_REF]. Proteins of the sugar phosphate cyclase family are key enzymes in the synthesis of C7 cyclitols. Enzymes of this family catalyse the cyclization of sugar compounds, an important step in primary and secondary metabolism [START_REF] Wu | A Comparative Analysis of the Sugar Phosphate Cyclase Superfamily Involved in Primary and Secondary Metabolism[END_REF]. Within this family, three main categories of enzymes use SH7P as a substrate: desmethyl-4-deoxygadusol synthase (DDGS), 2-epivaliolone synthase (EVS) and 2-epi-5-epi-valiolone synthase (EEVS), of which EEVS is the only known enzyme involved in C7N aminocyclitol synthesis [START_REF] Osborn | Evolution and Distribution of C 7 -Cyclitol Synthases in Prokaryotes and Eukaryotes[END_REF]. EEVS were originally only found in bacteria, where they catalyse the first step in the biosynthesis of C7N aminocyclitol secondary metabolites [START_REF] Mahmud | The C7N aminocyclitol family of natural products[END_REF][START_REF] Sieber | Isolation and Total Synthesis of Kirkamide, an Aminocyclitol from an Obligate Leaf Nodule Symbiont[END_REF]. More recently, EEVS homologs have been discovered in some Eukaryotes such as fish, reptiles, and birds as well [START_REF] Osborn | De novo synthesis of a sunscreen compound in vertebrates[END_REF][START_REF] Osborn | Evolution and Distribution of C 7 -Cyclitol Synthases in Prokaryotes and Eukaryotes[END_REF].

A second common feature of the leaf endophytes in Rubiaceae and Primulaceae is their reduced genomes. Leaf nodule Burkholderia symbionts of Rubiaceae and Primulaceae typically have smaller genomes than free-living relatives, as well as a lower coding capacity [START_REF] Pinto-Carbó | Evidence of horizontal gene transfer between obligate leaf nodule symbionts[END_REF]. This reductive genome evolution is thought to be a result of increased genetic drift sustained in bacteria that are strictly host-associated, which leads to fixation of deleterious and/or neutral mutations and eventually to the loss of genes [START_REF] Pettersson | Muller's ratchet in symbiont populations[END_REF]. This process is best documented in obligate insect symbionts such as Buchnera and Serratia, endosymbionts of aphids, or in Sodalis-allied symbionts of several insect groups [START_REF] Shigenobu | Genome sequence of the endocellular bacterial symbiont of aphids Buchnera sp[END_REF][START_REF] Toh | Massive genome erosion and functional adaptations provide insights into the symbiotic lifestyle of Sodalis glossinidius in the tsetse host[END_REF][START_REF] Manzano-Marín | A Freeloader? The Highly Eroded Yet Large Genome of the Serratia symbiotica Symbiont of Cinara strobi[END_REF]. Some of these symbionts have extremely small genomes and may present an extensive nucleotide bias towards adenosine and thymine (AT-bias) [START_REF] Moran | Genomics and Evolution of Heritable Bacterial Symbionts[END_REF]. The process of genome reduction has multiple stages: first, recently host-restricted symbionts begin accumulating pseudogenes and insertion elements [START_REF] Mccutcheon | Extreme genome reduction in symbiotic bacteria[END_REF][START_REF] Lo | Winding paths to simplicity: Genome evolution in facultative insect symbionts[END_REF][START_REF] Manzano-Marín | Snapshots of a shrinking partner: Genome reduction in Serratia symbiotica[END_REF]. Non-coding and selfish elements eventually get purged from the genomes over subsequent generations, which together with the general deletional bias in bacteria results in a decrease in genome size [START_REF] Mira | Deletional bias and the evolution of bacterial genomes[END_REF]. This ultimately leads to symbionts with tiny genomes, retaining only a handful of essential genes necessary for survival or performing their role in the symbiosis. This process has been well documented in the leaf nodule symbionts of Psychotria, Pavetta and Ardisia species, but little is known about the genomes and functions of endophytes in species that do not form leaf nodules, notably Rubiaceae species of the Vangueria and Fadogia genera.

Here, we performed a comparative study of Rubiaceae and Primulaceae leaf endophytes from leaf nodulating and non-nodulating plant species using genomes assembled from shotgun metagenome sequencing data as well as isolates. We constructed a dataset of 26 leaf symbiont genomes (17 of which from this study) from 22 plant species in 5 genera. All leaf symbionts show signs of genome reduction, in varying degree, and horizontal acquisition of secondary metabolite clusters is a universal phenomenon in these bacteria.

Material and Methods

Sample collection and DNA extraction

Leaves of Rubiaceae and Primulaceae species were freshly collected from different locations in South Africa or requested from the living collection of botanical gardens (Table S1).

Attempts to isolate the endophytes were made for all fresh samples collected in South Africa (Table S1). Leaf tissue was surface sterilized using 70% ethanol, followed by manual grinding of the tissue in 0.4% NaCl. Supernatants were plated on 10% tryptic soy agar medium (TSA, Sigma) and R2A medium (Oxoid) and incubated at room temperature for 3 days or longer until colonies appeared. Single colonies were picked and passaged twice on TSA medium.

Isolates were identified by PCR and partial sequencing of the 16S rRNA gene using the pA/pH primer pair (5'-AGAGTTTGATCCTGGCTCAG and 5'-AAGGAGGTGATCCAGCCGCA) [START_REF] Edwards | Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA[END_REF]. PCR products were sequenced using the Sanger method at Eurofins Genomics (Ebersberg, Germany). DNA was extracted from whole leaf samples as follows. Whole leaves were ground in liquid nitrogen using a mortar and pestle. Total DNA was extracted using the protocol of [START_REF] Inglis | Fast and inexpensive protocols for consistent extraction of high quality DNA and RNA from challenging plant and fungal samples for highthroughput SNP genotyping and sequencing applications[END_REF]. Total DNA from a Fadogia homblei isolate was extracted following Wilson [START_REF] Wilson | Preparation of Genomic DNA from Bacteria[END_REF]. Sequencing library preparation and 2x150 pairedend metagenome sequencing was performed by the Oxford Wellcome Centre for Human Genetics or by Novogene Europe (Cambridge, UK) using the Illumina NovaSeq 6000.

Sequencing reads were classified using Kraken v2.1.2 against a custom database comprising complete prokaryotic and plastid genome sequences deposited NCBI RefSeq (accessed 4/4/2021), and visualised using KronaTools v2.7.1 [START_REF] Ondov | Interactive metagenomic visualization in a Web browser[END_REF][START_REF] Wood | Improved metagenomic analysis with Kraken 2[END_REF].

Isolation of bacteria

Fresh leaf tissue was first washed in running tap water and surface-sterilized for 5 min in a 1.4% solution of sodium hypochlorite followed by 5 min in 70% ethanol. Leaves from a single plant were processed separately to prevent cross-contamination. Tissue was rinsed in sterile distilled water twice and ground using a sterile mortar and pestle in aseptic conditions.

Macerates were resuspended in 1 -5 mL of sterile 0.4% NaCl and serial dilutions were spread onto R2A agar [START_REF] Reasoner | A new medium for the enumeration and subculture of bacteria from potable water[END_REF] and 10% tryptic soy agar (10% TSA; 10% tryptic soy broth, Oxoid, Thermo Scientific, 18 g L-1 agar) and incubated at 28°C for a week.

Colonies were picked as they appeared, streaked out on TSA and incubated at room temperature. Strains were passaged three times on TSA prior to preservation at -80°C in tryptic soy broth supplemented with 20% glycerol.

Bacterial genome assembly

Sequencing reads were trimmed and filtered using fastp v0.21.0 with default settings, retaining reads with a minimum Phred score of 15 and less than 40% of bases failing the quality threshold [START_REF] Chen | fastp: an ultra-fast all-in-one FASTQ preprocessor[END_REF]. Overlapping paired-end reads were merged using NGmerge v0.3 with default settings [START_REF] Gaspar | NGmerge: merging paired-end reads via novel empirically-derived models of sequencing errors[END_REF]. Reads derived from isolates were assembled using Skesa v2.4.0 using default settings [START_REF] Souvorov | SKESA: strategic k-mer extension for scrupulous assemblies[END_REF]. Assembly statistics were compiled using Quast v5.1.0 [START_REF] Gundel | Vertically transmitted symbionts as mechanisms of transgenerational effects[END_REF]. For sequencing reads derived from new leaf samples, metagenome assemblies were created using metaSPAdes v3.15 on default settings but including the merged reads [START_REF] Nurk | metaSPAdes: a new versatile metagenomic assembler[END_REF]. Metagenomes were binned using Autometa v1.0.2, using a minimal contig length of 500 bp, taxonomy filtering (-m) and maximum-likelihood recruitment (using the -r option) [START_REF] Miller | Autometa: automated extraction of microbial genomes from individual shotgun metagenomes[END_REF].

Genome bins identified as Caballeronia, Paraburkholderia, or Burkholderia by Autometa were further assembled by mapping the original reads to these bins using smalt v0.7.6 [START_REF] Ponsting | SMALT -A New Mapper for DNA Sequencing Reads[END_REF]. Mapped reads were extracted using samtools v1.9 [START_REF] Li | The Sequence Alignment/Map format and SAMtools[END_REF] and reassembled using SPAdes v3.15 [START_REF] Bankevich | SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing[END_REF] in default settings but using the --careful option, and binned again using Autometa. Contigs likely derived from eukaryotic contamination were removed after identification by blastn searches (e-value < 1e -6 ) against the NCBI nucleotide database (accessed January 2021) [START_REF] Camacho | BLAST+: architecture and applications[END_REF]. Per-contig coverage information was calculated using samtools and contigs with less than 10% or more than 500% of the average coverage were manually investigated, and sequences likely derived from other bacterial or eukaryotic genomes were removed. The metagenome assembly approach was validated using the Fadogia homblei PRU 128010 dataset (Table S1) to compare the Paraburkholderia caledonica metagenome-assembled genome (MAG) to the genome sequence of strain Paraburkholderia caledonica R-82532 isolated from the same source material. MAG sequences of F. homblei endophytes contained 100% of the sequences of the R-82532 isolate genome, with only a small excess of contaminating sequences before manual filtering (MAG size = 8.90 Mb vs 8.71 Mb for the R-82532 assembly, with 100% average nucleotide identity on shared sequences).

To provide a more homogenous dataset for comparative genomics, Illumina read data for six previously published Rubiaceae symbionts, and the symbionts of Ardisia crenata and Fadogia homblei were re-assembled as above but using the published draft genomes as trusted contigs for both metaSPAdes and SPAdes assemblies (Table S2). The resulting assemblies were compared to the published assemblies using dotplots created by MUMmer v3.1 [START_REF] Marçais | MUMmer4: A fast and versatile genome alignment system[END_REF]. Genome assemblies of the symbionts of Psychotria kirkii [START_REF] Carlier | The eroded genome of a Psychotria leaf symbiont: hypotheses about lifestyle and interactions with its plant host[END_REF][START_REF] Carlier | Proteomics analysis of Psychotria leaf nodule symbiosis: improved genome annotation and metabolic predictions[END_REF] and Psychotria punctata [START_REF] Pinto-Carbó | Evidence of horizontal gene transfer between obligate leaf nodule symbionts[END_REF] were downloaded from Genbank (Table S2). To assess whether the (re-)assembled genomes or MAGs represent new species, genomes were analysed using TYGS (Type Strain Genome Server) (Meier-Kolthoff and Göker, 2019), and NCBI Blastn-based Average Nucleotide Identities (ANI) values calculated using the JSpecies web server, accessed Sept. 2021 [START_REF] Richter | JSpeciesWS: a web server for prokaryotic species circumscription based on pairwise genome comparison[END_REF] and the pyANI python package v0.2 (https://github.com/widdowquinn/pyani).

Genome annotation and pseudogene prediction

Assembled genomes were annotated using the online RASTtk pipeline [START_REF] Brettin | RASTtk: A modular and extensible implementation of the RAST algorithm for building custom annotation pipelines and annotating batches of genomes[END_REF], using GenemarkS as gene predictor, and locus tags were added using the Artemis software v18.1.0 [START_REF] Carver | Artemis: an integrated platform for visualization and analysis of high-throughput sequence-based experimental data[END_REF]. Prediction of pseudogenes was performed using an updated version of the pseudogene prediction pipeline previously used for leaf symbionts [START_REF] Carlier | Proteomics analysis of Psychotria leaf nodule symbiosis: improved genome annotation and metabolic predictions[END_REF]. Briefly, orthologs of predicted proteins sequences of each genome in a dataset of published Burkholderia genomes (Table S3) were determined using Orthofinder v2.5.2 [START_REF] Emms | OrthoFinder: Phylogenetic orthology inference for comparative genomics[END_REF] with default settings. The nucleotide sequences of each gene, including 200bp flanking regions (the query), were aligned to the highest scoring amino acid sequence in each orthogroup (the target) using TFASTY v3.6 [START_REF] Pearson | Flexible Sequence Similarity Searching with the FASTA3 Program Package[END_REF]. Genes were considered as pseudogenes if the alignment spanned over 50% of the query sequence and the query nucleotide sequence contained a frameshift, or a nonsense mutation resulting in an uninterrupted alignment shorter than 80% of the target sequence. Moreover, ORFs were classified as non-functional if at least one of the following criteria was true: amino acid sequence shorter than 50 residues which did not cluster in an orthogroup, and sequence without any significant blastx hit against the reference database (e-value cut off = 0.001); proteins without predicted orthologs in the Burkholderia dataset, but which showed a blastx hit against the reference set in an alternative reading frame; and finally proteins without any hit in the Burkholderia genome database or in the NCBI nr database. Blastx and blastp searches were performed using DIAMOND v2 [START_REF] Buchfink | Sensitive protein alignments at tree-of-life scale using DIAMOND[END_REF]. For the genomes of the symbionts of P. kirkii and P. punctata the original gene and pseudogene predictions were used. Insertion elements in both newly assembled and re-assembled genomes were predicted using ISEscan v1.7.2.3 with default settings [START_REF] Xie | ISEScan: automated identification of insertion sequence elements in prokaryotic genomes[END_REF].

Phylogenetic analysis

16S rRNA sequences were extracted from the endophyte (meta)genomes using Barrnap v0.9 (https://github.com/tseemann/barrnap). For genomes where no complete 16S rRNA could be detected, reads were mapped to the 16S rRNA gene of the closest relative with a complete 16S rRNA sequence. These reads were assembled using default SPAdes [START_REF] Prjibelski | Using SPAdes De Novo Assembler[END_REF] using the --careful option. Near complete (>95%) 16S rRNA sequences could be extracted using these methods, except for the hypothetical endophyte of Pavetta revoluta.

The 16S rRNA sequences were identified using the EzBiocloud 16S rRNA identification service (https://www.ezbiocloud.net/identify). Phylogenetic analysis of the leaf endophytes and Burkholderia s.l. genomes was performed using the UBCG pipeline v3.0 [START_REF] Na | UBCG: Up-to-date bacterial core gene set and pipeline for phylogenomic tree reconstruction[END_REF]. The pipeline was run using the default settings, except for the gap-cutoff (-f 80). The resulting superalignment of 92 core genes was used for maximum-likelihood phylogenetic analysis using RAxML v8.2.12, using the GTRGAMMA evolution model, and performing 100 bootstrap replications [START_REF] Stamatakis | RAxML version 8: A tool for phylogenetic analysis and post-analysis of large phylogenies[END_REF]. Plastid reference alignments were created using Realphy v1.12 using standard settings and the Coffea arabica chloroplast genome (NCBI accession NC_008535.1) as reference [START_REF] Bertels | Automated reconstruction of whole-genome phylogenies from short-sequence reads[END_REF] Core-genome phylogenies of symbiont genomes were constructed by individually aligning the protein sequences of all single-copy core genes using MUSCLE v3.8.1551, backtranslating to their nucleotide sequence using T-Coffee v13.45 [START_REF] Di Tommaso | T-Coffee: A web server for the multiple sequence alignment of protein and RNA sequences using structural information and homology extension[END_REF], and concatenating all nucleotide alignments into one superalignment using the AlignIO module of Biopython 1.78 [START_REF] Cock | Biopython: freely available Python tools for computational molecular biology and bioinformatics[END_REF]. Maximum-likelihood phylogenetic analysis was performed using RAxML, using the GTRGAMMA evolution model, 100 bootstrap replicates, and using partitioning to allow the model parameters to differ between individual genes. Phylogenetic trees were visualised and edited using iTOL [START_REF] Letunic | Interactive Tree Of Life (iTOL) v4: recent updates and new developments[END_REF].

Comparative genomics

Ortholog prediction between leaf symbiont genomes and a selection of reference genomes of the Burkholderia, Paraburkholderia and Caballeronia genera (BPC-set; selected using NCBI datasets tool (https://www.ncbi.nlm.nih.gov/datasets/genomes); Table S3) was performed using Orthofinder v2.5.2 using default settings [START_REF] Emms | OrthoFinder: Phylogenetic orthology inference for comparative genomics[END_REF].For the leaf endophytes, predicted pseudogenes were excluded from the analysis. The core genome of a certain group was defined as the number of orthogroups containing genes of all genomes in the group. Core genome overlap was visualised in Venn diagrams using InteractiVenn [START_REF] Heberle | InteractiVenn: a web-based tool for the analysis of sets through Venn diagrams[END_REF]. Non-essential core genes were identified by blastp searches against the database of essential genes (DEG) [START_REF] Zhang | DEG: a database of essential genes[END_REF], identifying as putative essential genes ORFs with significant matches in the database (e-value < 1e -6 ). Standardised functional annotation was performed using eggNOG-mapper v2. 1.2 (Huerta-Cepas et al., 2019;[START_REF] Cantalapiedra | eggNOG-mapper v2: Functional Annotation, Orthology Assignments, and Domain Prediction at the Metagenomic Scale[END_REF]. Enrichment of protein families in leaf symbiont genomes was determined by comparing the proportion of members of leaf symbionts and the BPC-set in orthogroups. Enriched KEGG pathways were identified by comparing the average pergenome counts of genes in every pathway between leaf symbiont genomes and genomes from the BPC-set. Presence of motility and secretion system clusters was investigated using the TXSScan models implemented in MacSyFinder [START_REF] Abby | MacSyFinder: A Program to Mine Genomes for Molecular Systems with an Application to CRISPR-Cas Systems[END_REF][START_REF] Abby | Identification of protein secretion systems in bacterial genomes[END_REF]. Homologues of the Ca. B. kirkii UZHbot1 putative 2-epi-5-epi-valiolone synthase (EEVS) were identified by blastp searches against the proteomes of the leaf symbiont genomes (e-value cut-off: 1e -6 ).

Putative EEVS genes were searched against the SwissProt database, and functional assignment was done by transferring the information from the closest match within the sugar phosphate cyclase superfamily [START_REF] Schneider | The Swiss-Prot protein knowledgebase and ExPASy: providing the plant community with high quality proteomic data and tools[END_REF][START_REF] Osborn | Evolution and Distribution of C 7 -Cyclitol Synthases in Prokaryotes and Eukaryotes[END_REF]. Contigs containing these genes were identified and extracted using Artemis, and aligned using Mauve [START_REF] Lòpez-Fernàndez | Whole-genome comparative analysis of virulence genes unveils similarities and differences between endophytes and other symbiotic bacteria[END_REF]. Gene phylogenies were constructed by creating protein alignments using MUSCLE followed by phylogenetic tree construction using FastTree v2.1.9 [START_REF] Price | FastTree: computing large minimum evolution trees with profiles instead of a distance matrix[END_REF], including the protein sequences of three closely related proteins in other species, determined by blastp searches against the RefSeq protein database (accessed July 2021). The data generated in this study have been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB52430 (https://www.ebi.ac.uk/ena.browser/view/PREJB52430).

GC-MS analysis of kirkamide

Extracts were derivatised with N-methyl-N-(trimethyl-silyl)-trifluoroacetamide (MSTFA, Merck Ltd) according to the method of Pinto-Carbó et al. (2016). Three replicates of the plant extracts were derivatised from a concentration of 1 mg/ml in 2 ml double distilled water as follows:

The extracts were filtered through 0.22 µm syringe fitted filters and 100 µl transferred to 2.0 ml screw top glass vials with 200 µl inserts and dried overnight under a nitrogen stream. The residues were dissolved in 50 µl MSTFA, vortexed for two minutes, left at 70 °C for one hour and then 50 µl pyridine was added as the solute. The derivatised samples were analysed on a Shimadzu GC-MS-QP2010 (Shimadzu Corporation, Japan) with ionization energy set at 70 eV.

The compounds were separated using a Rtx -5MS column (29.3 m x 250 µm x 0.25 µm i.d.; 0.25 µm df) with helium as the carrier gas. Splitless injections of 1 µl were performed, with the column flow set to linear velocity. Sampling time was set to 2 min, with the solvent cutoff time set to 3.5 min. The injector and interface temperatures were set at 250°C. The GC oven temperature program was set to an initial 40°C and held for 1 min, thereafter it was increased to 330°C at a rate of 7°C min -1 which was held for 10 min, bringing the total run time to 52 min. The MS ion source and interface temperatures were set to 250°C. The detector voltage was set to 0.1 kV, relative to the instrument tuning results. The mass-to-charge ratio (m/z) detection was set to start at 7 min (ensuring complete solvent elimination) and ranged from 45 to 650 m/z with a scan speed of 2 500 aum s -1 . Pyridine was used as a blank at the start of the analysis to observe any instrumental errors.

UPLC-QToF-MS analysis of streptol and streptol glucoside

The presence of underivatised streptol and streptol glucoside in the plant extracts was analysed using a Waters Synapt G2 high-definition mass spectrometry (HDMS) system (Waters Inc., Milford, Massachusetts, USA). The apparatus consists of a Waters Acquity UPLC connected to a quadropole-time-of-flight (QToF) instrument. The method of Georgiou et al.

(2021) was followed for the detection of streptol and streptol glcoside in negative mode [M-H] -. The samples were analysed using a Luna Omega 1.6 µm C18 100 A, 100 x 2.1 mm (Phenomenex, Separations) column and a solvent system that consisted of MeCN:H2O (A, 8:2, 0.1 % NH4OAc) and MeCN:H2O (B, 2:8, 0.1 % NH4OAc). The gradient was set to start at 95 % of B and to decrease to 50 % of B in 7 min, for the next 2 min the gradient was kept at 50 % of B, the gradient was then gradually decreased from 50 % to 5 % of B for the next 3 min and was followed by a column wash for the next 2 min giving a total run time of 12 min. The column temperature was 40 °C, injection volume 7 µl and the flow rate 0.3 ml min -1 . Mass to charge ratios (m/z) were recorded between 50 and 1 200 Da. High energy collision induced dissociation (CID) was used for tandem MS fragmentation. The collision energy for the ramping was set to increase from 10 V to 20 V in order to get a range of data. The full scan MS data was recorded from the QTOF-MS and XICs (extracted ion chromatograms) were used for processing the data to single out the ions of interest. In some instances targeted MS/MS spectra were employed for the detection of streptol. Presence of streptol was determined by the presence of spectral features with ion fragments at 85, 108, 111, 121 and 175 m/z and a monoisotopic mass of 175.06119 (± 5 ppm). Presence of streptol-glucoside was determined by the presence of spectral features with ion fragments at 112, 139, 175 and 337 m/z and a monoisotopic mass of 337.1140 (± 5 ppm).

Results

Detection and identification of leaf endophytes

To gain insight into potential association of various Primulaceae and Rubiaceae species with Burkholderia s.l. endosymbionts, we collected samples from 16 Rubiaceae (1 Fadogia sp., 5 Pavetta spp., 2 Psychotria spp., and 8 Vangueria spp.) and 3 Primulaceae (3 Ardisia spp.) species (Table S1). We extracted DNA from entire leaves and submitted the samples to shotgun sequencing without pre-processing of the samples to remove host or organellar DNA. We found evidence for endophytic Burkholderia in 14 out of 19 species investigated (Table S1). In these samples, the proportion of sequencing reads identified as Burkholderiaceae ranged from 5% to 57% of the total, except for the Pavetta revoluta sample (0.4%) and 1 of 2 Vangueria infausta samples (0.9%). Analysis of 16S rRNA sequences revealed 100% pairwise identity over 1529 bp suggesting that the same endophyte species was present in both V. infausta samples. In Pavetta revoluta, the closest relative of the leaf endophyte based on 16S rRNA sequence similarity was Caballeronia calidae (98.89% identity over 808 bp; Table S1). Of the nine species with significant amounts of Burkholderia s.l. reads and for which isolation attempts were made (Table S1), only the endophyte of Fadogia homblei could be cultured (isolate R-82532). Leaf samples of four species (Psychotria capensis, Psychotria zombamontana, Pavetta ternifolia, and Pavetta capensis) contained low amounts of bacterial DNA (<2% of reads), and likely do not have stable symbiotic endophyte associations. Seven percent of the reads obtained from the Pavetta indica sample were classified as bacterial, but with a diverse range of taxa present indicating possible contamination with surface bacteria (Figure S1). Plastid phylogenies indicated that samples attributed to Pavetta capensis and Pavetta indica did not cluster with other Pavetta species (Figure S2). Analysis of genetic markers revealed that our Pavetta indica sample was likely a misidentified Ixora species. Analysis of Pavetta capensis marker genes revealed the specimen is likely part of the Apocynaceae plant family, with a 100% identity match against the rbcL sequence of Pleiocarpa mutica. These samples were not taken into account in further analyses.

Analysis of the 16S rRNA sequences extracted from metagenome-assembled genomes (MAGs) identified all leaf endophytes as Burkholderia s.l. (Table S1). Phylogenetic analysis shows that all endophytes of Psychotria, Pavetta, and Ardisia cluster within the genus Caballeronia, while the endophytes of Vangueria and Fadogia belong to the Paraburkholderia genus (Figure 1A). All endophytes of Ardisia are closely related to each other and form a clade with Caballeronia udeis and Caballeronia sordidicola. Based on the commonly used ANI (95-96%) cut-off [START_REF] Richter | Shifting the genomic gold standard for the prokaryotic species definition[END_REF], these endophytes are separate species from C. udeis and C. sordidicola (ANI <94%; 16S rRNA sequence identity <98.4). The endophytes of Ardisia crenata and Ardisia virens are very closely related and belong to the same species: Ca. Burkholderia crenata (ANI >99%; 16S rRNA sequence identity 99.8%) (Table S4). Similarly, the endophytes of Ardisia cornudentata and Ardisia mamillata belong to the same species (ANI = 95.56%), which we tentatively named Ca.

Caballeronia ardisicola (species epithet from Ardisia, the genus of the host species, and the Latin suffix -cola (from L. n. incola), dweller, see species description in Supplementary Information). Endophytes of Psychotria and Pavetta are scattered across the Caballeronia phylogeny, but all are taxonomically distinct from free-living species (Figure 1A; ANI <93% with closest non-endophyte relatives). Each of these endophytes also represents a distinct bacterial species with pairwise Average Nucleotide Identity (ANI) values below the commonly accepted species threshold of 95-96%, including the endophyte of Pavetta hochstetteri which we tentatively named Candidatus Caballeronia hochstetteri (Table S4 and Supplementary information). Although their MAGs share 95.65% ANI (a borderline value for species delineation), Ca. B. schumanniana (endophyte of Pavetta schumanniana) and Ca. B. kirkii have been previously described as distinct species on the basis of 16S rRNA gene sequence similarity [START_REF] Verstraete | Endophytic Bacteria in Toxic South African Plants: Identification, Phylogeny and Possible Involvement in Gousiekte[END_REF] (Table S4). The endophytes of Vangueria and Fadogia form three distinct lineages of Paraburkholderia. The endophytes of Vangueria dryadum and Vangueria macrocalyx are nearly identical (ANI >99.9%; identical 16S rRNA), but do not belong to any known Paraburkholderia species (ANI <83% with closest relative Paraburkholderia species). We tentatively assigned these bacteria to a new species which we named Ca. Paraburkholderia dryadicola (from a Dryad, borrowed from the species epithet of one of the host species, and Latin suffix -cola, see species description in Supplementary Information). Similarly, the endophytes of V. infausta, V. esculenta, V. madagascariensis, V. randii, and V. soutpansbergensis cluster together with Paraburkholderia phenoliruptrix (Figure 1A). While the endophyte of Vangueria soutpansbergensis forms a separate species (named here Ca. Paraburkholderia soutpansbergensis; ANI <95% with P. phenoliruptrix) the other endophytes fall within the species boundaries of P. phenoliruptrix. (ANI 95-96% between these endophytes and P. phenoliruptrix). Lastly, the endophytes of Fadogia homblei and Vangueria pygmaea showed identical 16S rRNA sequences, and clustered with Paraburkholderia caledonica, P. strydomiana, and P. dilworthii (Figure 1A). Similarly high ANI values (>97.5%) and 16S rRNA sequence similarity (>99.7%) ambiguously fall within the species boundaries of both P. caledonica and P. strydomiana. Because endophytes of F. homblei were previously classified as P. caledonica [START_REF] Verstraete | Endophytic Bacteria in Toxic South African Plants: Identification, Phylogeny and Possible Involvement in Gousiekte[END_REF][START_REF] Verstraete | Intraspecific variation in Burkholderia caledonica: Europe vs. Africa and soil vs. endophytic isolates[END_REF], we propose classifying the endophytes of F. homblei and V. pygmaea as members of P. caledonica, and consider P. strydomiana a later heterotypic synonym of P. caledonica.

Phylogenetic analysis based on the core genomes of endophytes indicates a general lack of congruence with the host plant phylogeny (Figure S3). Endophytes of Ardisia are monophyletic within the Caballeronia genus and follow the host phylogeny. In contrast, endophytes of Pavetta are not monophyletic and are nested within the Psychotria endophytes. Similarly, the Fadogia homblei endophyte clusters with endophytes of Vangueria.

Leaf endophyte genomes show signs of genome reduction.

We could assemble nearly complete bacterial genomes for all samples where we detected Burkholderia endophytes, except for those of the Pavetta revoluta and one Vangueria infausta sample with too few bacterial reads. Binning analysis grouped endophyte sequences in a single bin per sample, with high completeness and purity (Table 1). Most assemblies ranged between 3.5 and 5 Mbp in size, with 2 outliers: 2.58 Mbp for Ca. B. crenata Avir (the endophyte of Ardisia virens), and 8.92 Mbp for P. caledonica R-49542 (endophyte of Fadogia homblei)(Table 1). The G+C-content of all MAGs fell in the range of 59-64 percent G+C content, which is within the range of free-living Paraburkholderia and Caballeronia genomes [START_REF] Vandamme | Comparative Genomics of Burkholderia singularis sp. nov., a Low G+C Content, Free-Living Bacterium That Defies Taxonomic Dissection of the Genus Burkholderia[END_REF]. All MAGs showed signs of ongoing genome reduction. Because of rampant null or frameshift mutations, a large proportion of predicted CDS code for non-functional proteins. As a result, coding capacity is low for all endophyte MAGs varying between 83% in P. caledonica R-49542 (Figure 1B, Figure S4) and 40% in Ca. C. ardisicola Acor, the endophyte of Ardisia cornudentata (Figure 1B, Figure S5). In addition, insertion sequence (IS) elements make up a large amount of the MAGs: 1.97% of the assembly size on average, but up to almost 10% in some symbionts of Psychotria (Table 1).

Reassembly of previously investigated endophytes of Psychotria and Pavetta yielded assemblies of similar size to the original assemblies, except for Ca. Burkholderia schumanniana UZHbot8 (endophyte of Pavetta schumanniana). The original genome assembly size was estimated at 2.4 Mbp, while our reassembly counted 3.62 Mbp. A dot plot between both assemblies indicated that the size discrepancy is not solely due to differential resolution of repeated elements (Figure S6). Thus, our new assembly includes 1.2 Mbp of genome sequence that was missed in the original assembly.

Burkholderia leaf endophytes in Rubiaceae and Primulaceae shared a core genome of 607 genes (Figure S7). Even within specific phylogenetic lineages the core genomes were small: 774 genes in endophytes belonging to the Caballeronia symbionts of Psychotria and Pavetta, 1001 genes in endophytes of Caballeronia symbionts of Ardisia, and 1199 in Paraburkholderia endophytes of Fadogia and Vangueria. This corresponds to 29.5%, 52.4%, and 28.4% of the average functional proteome for each species cluster, respectively. Only 28 proteins of the total core genome did not show significant similarity with proteins from the database of essential genes (Table S5). Eleven of these proteins have unknown functions and five are membrane-related. Fifteen genes of the endophyte core genome did not have orthologs in >95% of related Burkholderia, Caballeronia, and Paraburkholderia genomes (Table S6). No COG category was specifically enriched in this set of proteins.

Because secretion of protein effectors is often a feature of endophytic bacteria [START_REF] Brader | Ecology and Genomic Insights into Plant-Pathogenic and Plant-Nonpathogenic Endophytes[END_REF], we searched for genes encoding various secretion machineries in the genomes of Burkholderia endophytes. Flagellar genes, as well as Type III, IV or VI secretion system were not conserved in all leaf endophytes (Figure S8). macrocalyx, respectively). The flagellar apparatus is missing in both Ca. P. dryadicola MAGs, in Ca. P. soutpansbergensis Vsou, and in P. phenoliruptrix Vesc (the endophyte of V. esculenta), and is incomplete in some other P. phenoliruptrix endophytes. Lastly, only the genomes of Paraburkholderia caledonica endophytes R-49542 and R-82532 encode a complete set of core Type VI secretion system proteins.

Genes related to cyclitol metabolism are enriched in leaf endophytes

We wondered if specific metabolic pathways might be enriched in genomes of leaf symbionts, despite rampant reductive evolution. We assigned KEGG pathway membership for each predicted functional CDS (thus excluding predicted pseudogenes) in leaf symbiont genomes or MAGs as well as a set of free-living representative Paraburkholderia or Caballeronia species. The number of genes assigned to a majority of the KEGG pathways (256 pathways in total) was significantly smaller in endophyte genomes compared to their free-living relatives. A small portion (86 pathways) did not differ between leaf symbionts and free-living representatives. Genes belonging to a single pathway were significantly enriched in leaf endophytes: acarbose and validamycin biosynthesis (KEGG pathway map00525).

Acarbose and validamycin are aminocyclitols synthesized via 2-epi-5-epi-valiolone synthase (EEVS). EEVS catalyses the first committed step of C7N aminocyclitol synthesis [START_REF] Mahmud | The C7N aminocyclitol family of natural products[END_REF][START_REF] Mahmud | Progress in aminocyclitol biosynthesis[END_REF], and likely plays a role in the production of kirkamide, a natural C7N aminocyclitol present in leaves of Psychotria kirkii and other nodulated Rubiaceae, as well as streptol and streptol glucoside, 2 cyclitols with herbicidal activities [START_REF] Pinto-Carbó | Evidence of horizontal gene transfer between obligate leaf nodule symbionts[END_REF]. Indeed, of 10 Ca. Burkholderia kirkii UZHbot1 genes assigned to KEGG pathway map00525, 8 genes were previously hypothesised to play a direct role in the synthesis of C7N aminocyclitol or derived compounds [START_REF] Pinto-Carbó | Evidence of horizontal gene transfer between obligate leaf nodule symbionts[END_REF]. Similarly, 7 out of 11 orthogroups most enriched in leaf endophytes contained a gene putatively involved in cyclitol synthesis in Ca. Burkholderia kirkii UZHbot1 (Table S7) [START_REF] Carlier | The eroded genome of a Psychotria leaf symbiont: hypotheses about lifestyle and interactions with its plant host[END_REF][START_REF] Sieber | Isolation and Total Synthesis of Kirkamide, an Aminocyclitol from an Obligate Leaf Nodule Symbiont[END_REF]. To gain a better understanding of the distribution of cyclitol biosynthetic clusters in leaf endophytes, we searched for homologs of the two 2-epi-5-epi-valiolone synthase (EEVS) genes of Ca. Burkholderia kirkii UZHbot1 (locus tags BKIR_C149_4878 and BKIR_C48_3593) in the other leaf endophyte genomes. We detected putative EEVS homologs in all but the two genomes of Ca. B. crenata. For Ca. B. crenata UZHbot9 we have previously shown the genome encodes a non-ribosomal peptide synthase likely responsible for the synthesis of the depsipeptide FR900359 [START_REF] Fujioka | Structure of FR900359, a cyclic depsipeptide from Ardisia crenata sims[END_REF][START_REF] Carlier | The genome analysis of Candidatus Burkholderia crenata reveals that secondary metabolism may be a key function of the Ardisia crenata leaf nodule symbiosis[END_REF][START_REF] Crüsemann | Heterologous Expression, Biosynthetic Studies, and Ecological Function of the Selective Gq-Signaling Inhibitor FR900359[END_REF], and these genes were also detected in Ca. B. crenata Avir. Because

Evolution of cyclitol metabolism in leaf endophytic Burkholderia

Phylogenetic analysis of the endophyte EEVS protein sequences showed the presence of two main clades of Burkholderia EEVS homologs, as well as a divergent homolog in the genome of Ca. C. ardisicola Acor, and a second divergent homolog in Ca. P. dryadicola Vdry and Vmac (Figure 2A). The gene context of these EEVS genes in the different clades reveals that the two main EEVS clades correspond to the two conserved gene clusters previously hypothesized to play a role in kirkamide and streptol glucoside biosynthesis in Ca.

Burkholderia kirkii [START_REF] Carlier | Proteomics analysis of Psychotria leaf nodule symbiosis: improved genome annotation and metabolic predictions[END_REF]. The gene order of these clusters is very similar in every genome, with a similar genomic context in closely related genomes (Tables 2 &3 andFigure S10). These gene clusters are generally flanked by multiple mobile elements, consistent with acquisition via horizontal gene transfer (Table S9). Furthermore, the EEVS phylogeny did not follow the species phylogeny, indicating that HGT or gene conversion occurred (Figure 2A and Figure 2B). For clarity, we named the two main putative cyclitol biosynthetic gene clusters S-cluster (for streptol) and K-cluster (for kirkamide) based on previous biosynthetic hypotheses from in silico analysis of the putative cyclitol gene clusters of Ca. B. kirkii (Figure 2A) [START_REF] Pinto-Carbó | Evidence of horizontal gene transfer between obligate leaf nodule symbionts[END_REF]. Both K and S-clusters encode a core set of proteins linked to sugar analog biosynthesis: a ROK family protein and a HAD family hydrolase, and both contain aminotransferases (although from different protein families).

Two EEVS genes contain nonsense mutations and are likely not functional: the S-cluster EEVS of Ca. Burkholderia humilis UZHbot5, and the K-cluster EEVS of Ca. Burkholderia brachyanthoides UZHbot7. The MAG of Ca. B. humilis UZHbot5 still contains an apparently functional K-cluster EEVS, while the pseudogenized EEVS of Ca. B. brachyanthoides UZHbot7 is the only homolog in the MAG. Interestingly, genes of the K-cluster appear to be exclusive to Psychotria and Pavetta symbionts, while the S-cluster is more widespread, including in the MAGs of Vangueria endophytes. Accordingly, we detected kirkamide in leaf extracts of Psychotria kirkii, but in none of the Fadogia or Vangueria species we tested (see supplementary information). We also detected signals that were consistent with streptol/valienol and streptol glucoside by UPLC-QToF-MS in all samples. However, these signals occurred in a noisy part of the chromatogram, and we cannot confidently conclude if these m/z features come from a single streptol derivative or from several compounds. The MAG of Ca. P. soutpansbergensis Vsou and genomes of P. caledonica R-49542 and R-82532 encoded EEVS homologs of the K-cluster, but the full complement of the genes of the K-cluster is missing (Table 3 andFigure S10). In both cases the EEVS gene is flanked by IS elements (Table S9). Accordingly, we did not detect kirkamide in leaf samples from either Fadogia homblei or V. soutpansbergensis in our chemical analyses. The MAGs of Ca. P. dryadicola Vmac and Vdry encodes an EEVS that clusters outside of the K-and S-EEVS clusters. Genes with putative functions similar to those of the K-cluster are located in the vicinity of the EEVS in the MAGs of both Ca. P. dryadicola strains: oxidoreductases, an aminotransferase, and an N-acetyltransferase (Table S8). Similarly, Ca. C. ardisicola Acor contains a second divergent EEVS, in addition to the S-cluster EEVS. This EEVS belongs to a larger gene cluster coding for similar functions also found in the other EEVS-clusters, but contains at least one frameshift mutation and no longer codes for a functional enzyme (Table S8). Lastly, Ca. B. verschuerenii UZHbot4 contains a second, recently diverged EEVS paralog of the K-cluster. This EEVS is part of a small cluster of genes, with putative functions divergent from those found in the other EEVS-clusters and likely does not play a role in kirkamide synthesis (Table S8). with EEVS similar to the K-cluster. K* -Only the K-cluster EEVS is present, not the accessory genes.

Discussion

Different evolutionary origins of leaf symbioses in different plant genera

In this work, we investigated the evolution of associations between Burkholderia s. l. bacteria and plants of the Rubiaceae and Primulaceae families, and attempted to identify key characteristics of these associations. To this end, we re-analyzed publicly available genome data from previous research, and sequenced and assembled the genomes of an additional 17 leaf endophytes. In addition to leaf endophytes which had been previously detected (Lemaire, Smets, et al., 2011;[START_REF] Verstraete | Endophytic Bacteria in Toxic South African Plants: Identification, Phylogeny and Possible Involvement in Gousiekte[END_REF][START_REF] Verstraete | Symbiotic ß-Proteobacteria beyond Legumes: Burkholderia in Rubiaceae[END_REF][START_REF] Ku | Phylogenetic and Cophylogenetic Analyses of the Leaf-Nodule Symbiosis in Ardisia Subgenus Crispardisia (Myrsinaceae): Evidence from Nuclear and Chloroplast Markers and Bacterial rrn Operons[END_REF], we document here the presence of Burkholderia s.l. symbionts in Pavetta hochstetteri and Vangueria esculenta, and possibly Pavetta revoluta. In contrast to previous findings [START_REF] Lemaire | Screening for leaf-associated endophytes in the genus Psychotria (Rubiaceae)[END_REF], we could not detect evidence of leaf endophytes in Psychotria capensis, but did confirm the absence of leaf endophytes in Psychotria zombamontana. Phylogenetic placement of hosts and endophytes are consistent with previous data, except for the placement of Vangueria macrocalyx and its endophyte [START_REF] Lemaire | Screening for leaf-associated endophytes in the genus Psychotria (Rubiaceae)[END_REF][START_REF] Verstraete | Symbiotic ß-Proteobacteria beyond Legumes: Burkholderia in Rubiaceae[END_REF]. Both chloroplast sequences of V. macrocalyx and V. dryadum and the MAGs of their endophytes were nearly identical while previous research showed a clear phylogenetic difference both between the host species and their endophytes [START_REF] Verstraete | Symbiotic ß-Proteobacteria beyond Legumes: Burkholderia in Rubiaceae[END_REF]. Blastn analysis of plant genetic markers (ITS, petB, rpl16, trnTF) of both species against the NCBI nr database showed higher identities to markers from Vangueria dryadum than to those of Vangueria macrocalyx. However, since comparison of the vouchered V. macrocalyx specimen to other vouchered Vangueria dryadum and V. macrocalyx by expert botanists clearly separated both species, we decided to consider both species distinct.

Previous studies showed that Rubiaceae and Primulaceae species with heritable leaf symbionts are monophyletic within their respective genera [START_REF] Lemaire | Bacterial Leaf Symbiosis in Angiosperms: Host Specificity without Co-Speciation[END_REF][START_REF] Verstraete | Symbiotic ß-Proteobacteria beyond Legumes: Burkholderia in Rubiaceae[END_REF]. Thus, while the transition to a symbiotic state arose separately in multiple plant genera, it likely evolved only once in each plant genus. The only exception is the Psychotria genus, where it likely arose twice: once in species forming leaf nodules, and once in species without leaf nodules [START_REF] Lemaire | Screening for leaf-associated endophytes in the genus Psychotria (Rubiaceae)[END_REF]. The repeated emergence of leaf symbiosis is reflected on the microbial side as well. A parsimonious interpretation of whole genome phylogenetic analyses indicates that Burkholderia endophytes evolved independently at least 8 times, most probably from ancestors with an environmental lifestyle (Figure 1A). Caballeronia endophytes of Ardisia seem to have emerged once, with most closely related species commonly isolated from soil [START_REF] Lim | Burkholderia sordidicola sp. nov., isolated from the white-rot fungus Phanerochaete sordida[END_REF][START_REF] Vandamme | Burkholderia humi sp. nov., Burkholderia choica sp. nov., Burkholderia telluris sp. nov., Burkholderia terrestris sp. nov. and Burkholderia udeis sp. nov.: Burkholderia glathei-like bacteria from soil and rhizosphere soil[END_REF][START_REF] Uroz | Caballeronia mineralivorans sp. nov., isolated from oak-Scleroderma citrinum mycorrhizosphere[END_REF]. As previously reported, symbionts of Psychotria and Pavetta cluster in 3 distinct phylogenetic groups within the Caballeronia genus. Finally, symbionts of Vangueria and Fadogia belong to 5 distinct clades within the genus Paraburkholderia. Apart from Ca. P. dryadicola that is without closely related isolates, endophytic Paraburkholderia species also cluster together with species commonly isolated from soil [START_REF] Verstraete | Intraspecific variation in Burkholderia caledonica: Europe vs. Africa and soil vs. endophytic isolates[END_REF][START_REF] Beukes | Paraburkholderia strydomiana sp. nov. and Paraburkholderia steynii sp. nov.: rhizobial symbionts of the fynbos legume Hypocalyptus sophoroides[END_REF]. High hostspecificity is a hallmark of the Psychotria, Pavetta, and Ardisia leaf symbiosis, but this characteristic is not shared in Vangueria and Fadogia. Based on genome similarity, we identified at least three phylogenetically divergent endophyte species that can infect multiple hosts: P. caledonica, P. phenoliruptrix, and Ca. P. dryadicola. It is also possible that these plants are in the early stages of endophyte capture, where the plant is open to acquire endophytes from the soil, as previously hypothesized for F. homblei [START_REF] Verstraete | Symbiotic ß-Proteobacteria beyond Legumes: Burkholderia in Rubiaceae[END_REF].

Endophytes might later evolve to become host-restricted and vertically transmitted, leading to diversification from their close relatives and forming new species. This could, for example, already be the case for Ca. P. soutpansbergensis, which is related to P. phenoliruptrix but shows a more divergent genome (ANI <95%). Overall, these results highlight the general plasticity of bacteria in the Burkholderia s.l., as well as the probable frequent occurrence of host-switching or horizontal transmission within leaf symbiotic associations.

Genome reduction is a common trait of leaf endophytes

Bacterial genomes contain a wealth of information yet few leaf endophyte genomes are available. In this study we provide an additional thirteen leaf endophyte genome assemblies among which the first genomes of endophytes from Vangueria and Fadogia. Aside from the genomes of P. caledonica endophytes, all leaf endophyte genomes were small, mostly between 3.5 and 5 Mbp. This is well below the average 6.85 Mbp of the Burkholderiaceae family [START_REF] Carlier | The genome analysis of Candidatus Burkholderia crenata reveals that secondary metabolism may be a key function of the Ardisia crenata leaf nodule symbiosis[END_REF][START_REF] Pinto-Carbó | Evidence of horizontal gene transfer between obligate leaf nodule symbionts[END_REF]. In addition to their small sizes, the genomes of Psychotria, Pavetta, and Ardisia endophytes show signs of advanced genome reduction. Only 41-70% of these genomes code for functional proteins, compared to an average of about 90% for free-living bacteria [START_REF] Land | Insights from 20 years of bacterial genome sequencing[END_REF]. Most of these genomes also contain a high proportion of mobile sequences, up to 9% of the total assembly. Together, this indicates ongoing reductive genome evolution, a process often observed in obligate endosymbiotic bacteria [START_REF] Moran | Genomic changes following host restriction in bacteria[END_REF][START_REF] Bennett | Heritable symbiosis: The advantages and perils of an evolutionary rabbit hole[END_REF]. Interestingly, the MAGs of Vangueria and Fadogia endophytes, which are not contained in leaf nodules, also show signs of genome erosion: most MAGs of P. phenoliruptrix endophytes are at or below 5 Mbp in size, with over half of their proteome predicted as non-functional. The genomes of 2 Ca. P. dryadicola strains even approach the level of genome reduction found in most Psychotria symbionts. The intermediate genome reduction in endophytes of Vangueria and Fadogia could be explained by the relatively recent origin of the symbiosis, although leaf symbiosis in Fadogia has been estimated to be older than in Vangueria (7.6 Mya vs. 3.7 Mya) [START_REF] Verstraete | Non-nodulated bacterial leaf symbiosis promotes the evolutionary success of its host plants in the coffee family (Rubiaceae)[END_REF]. Other factors likely contribute to the extent or pace of genome reduction in the endophytes, such as mode of transmission and transmission bottlenecks.

The larger genome size and fewer pseudogenes compared to most other leaf endophytes may explain why we could isolate P. caledonica endophytes from F. homblei, but not other endophytes. We could not identify essential genes or pathways that were consistently missing in the genomes or MAGs of Burkholderia endophytes. It is therefore possible that other endophytic bacteria may be culturable using more complex or tailored culture conditions.

Secondary metabolism as key factor in the evolution of leaf symbiosis

Although leaf symbionts share a similar habitat and all belong to the Burkholderia s. l., their core genome is surprisingly small and consists almost entirely (95%) of genes that are considered essential for cellular life. This poor conservation of accessory functions perhaps reflects the large diversity and possible redundancy of functions encoded in the genomes of Burkholderia s.l. that associate with plants. Interestingly, the capacity for production of secondary metabolites is a key common trait of Burkholderia leaf endophytes. We previously showed that Ca. B. crenata produces FR900359, a cyclic depsipeptide isolated from A. crenata leaves [START_REF] Carlier | The genome analysis of Candidatus Burkholderia crenata reveals that secondary metabolism may be a key function of the Ardisia crenata leaf nodule symbiosis[END_REF]. This non-ribosomal peptide possesses unique pharmacological properties and may contribute to the protection of the host plant against insects [START_REF] Carlier | The genome analysis of Candidatus Burkholderia crenata reveals that secondary metabolism may be a key function of the Ardisia crenata leaf nodule symbiosis[END_REF][START_REF] Crüsemann | Heterologous Expression, Biosynthetic Studies, and Ecological Function of the Selective Gq-Signaling Inhibitor FR900359[END_REF]. However, our data suggests that the production of cyclitols is widespread in leaf endophytic Burkholderia. Indeed, with the exception of Ca. B. crenata cited above, we found evidence for the presence of cyclitol biosynthetic pathways in all genomes of leaf endophytic Burkholderia. We have previously reported the presence of two gene clusters containing a 2-epi-5-epi-valiolone synthase (EEVS) in MAGs of Psychotria and Pavetta symbionts [START_REF] Pinto-Carbó | Evidence of horizontal gene transfer between obligate leaf nodule symbionts[END_REF]. These gene clusters are likely responsible for the production of 2 distinct cyclitols: kirkamide, a C7N aminocyclitol with insecticidal properties which has been detected in several Psychotria plants; and streptol-glucoside, a plant-growth inhibitor likewise detected in Psychotria kirkii [START_REF] Sieber | Isolation and Total Synthesis of Kirkamide, an Aminocyclitol from an Obligate Leaf Nodule Symbiont[END_REF][START_REF] Pinto-Carbó | Evidence of horizontal gene transfer between obligate leaf nodule symbionts[END_REF][START_REF] Hsiao | Synthesis and Biological Evaluation of the Novel Growth Inhibitor Streptol Glucoside, Isolated from an Obligate Plant Symbiont[END_REF]. EEVS from leaf symbionts belong to four phylogenetic clusters, including the two EEVS genes previously detected in Psychotria and Pavetta symbionts [START_REF] Pinto-Carbó | Evidence of horizontal gene transfer between obligate leaf nodule symbionts[END_REF]. Similar to these previously analysed leaf endophyte genomes, the EEVS gene clusters in the newly sequenced genomes are flanked by IS-elements, and their phylogeny is incongruent with the species phylogeny. This indicates that these genes and clusters are likely acquired via horizontal gene transfer. This hypothesis is strengthened by the fact that the closest homologs of the genes in the EEVS clusters are found in genera as diverse as Pseudomonas, Streptomyces, and Noviherbaspirillum, but are rare in the genomes of Burkholderia s.l. The presence of the two main EEVS gene clusters (K-cluster and S-cluster) is not strictly linked to the symbiont or host taxonomy. For example, the EEVS of the K-cluster (hypothesised to produce kirkamide) is present in all sequenced symbionts of Psychotria and Pavetta but also in the endophytes of F. homblei and V. soutpansbergensis. However, in the latter two, accessory genes of the Kcluster are absent. It is possible that this EEVS interacts with gene products of other secondary metabolite clusters [START_REF] Osborn | Evolution and Distribution of C 7 -Cyclitol Synthases in Prokaryotes and Eukaryotes[END_REF]. We also noticed that some endophyte MAGs contain multiple EEVS genes or gene clusters. This could provide functional redundancy, protecting against the rampant genome erosion. For example, two genes of the S-cluster Ca. C. hochstetteri PhocE (endophyte of Pavetta hochstetteri) are likely pseudogenes, while the K-cluster gene is still complete. On the other hand, in Ca.

Burkholderia humilis UZHbot5 (endophyte of Psychotria humilis) seven out of ten genes of the S-cluster (including the EEVS) are either missing or non-functional, and the K-cluster is heavily reduced with only four functional genes out of eight (including the EEVS). As one functional EEVS copy remains, it is possible that genes located elsewhere in the genome provide these functions, as kirkamide has previously been detected in extracts of P. humilis [START_REF] Pinto-Carbó | Evidence of horizontal gene transfer between obligate leaf nodule symbionts[END_REF]. Alternatively, this symbiosis may have reached a "point of no return" where host and symbiont have become dependent on each other and nonperforming symbionts can become fixed in the population [START_REF] Bennett | Heritable symbiosis: The advantages and perils of an evolutionary rabbit hole[END_REF].

The presence of gene clusters coding for specialised secondary metabolites in all leaf symbionts could indicate that secondary metabolite production is either a prerequisite for or a consequence of an endophytic lifestyle. The fact that P. caledonica leaf symbionts have EEVS genes of different origin favours the hypothesis that the acquisition of secondary metabolism precedes an endophytic lifestyle. In this case, the ancestor of both endophytes may have acquired differing EEVS genes or EEVS gene clusters through HGT followed by infection of the respective host plants. The lack of EEVS homolog in Ca. B. crenata Avir and Acre indicates that production of cyclitols is not essential for leaf symbiosis. Interestingly, MAGs of the sister species Ca. C. ardisicola Amam and Acor encode an EEVS and the full Scluster complement. Since there is strong phylogenetic evidence of co-speciation in the Burkholderia/Ardisia association (Lemaire, Smets, et al., 2011;[START_REF] Ku | Phylogenetic and Cophylogenetic Analyses of the Leaf-Nodule Symbiosis in Ardisia Subgenus Crispardisia (Myrsinaceae): Evidence from Nuclear and Chloroplast Markers and Bacterial rrn Operons[END_REF] plants [START_REF] Eberl | Members of the genus Burkholderia: good and bad guys[END_REF], and many of these associations may be transient in nature. However, useful traits such as synthesis of protective metabolites may help stabilise these relationships, resulting in long-term associations such as leaf symbiosis.

Author contributions:

AC, MM, and BD designed the research. MM identified and collected wild plant specimens from the Pretoria region (South Africa). BD, MB, SS, and AC performed the laboratory experiments and analyses. GM analysed metabolomics data. PV analysed data and made taxonomic assignments. BD, MM and AC wrote the manuscript with input from all authors. Table 2: EEVS S-cluster organisation in endophyte genomes. Genomes of the same host with the same cluster layout are merged. X: Gene present; -: Gene absent; Ψ: Gene predicted to be pseudogene; *: genes present on a different contig than the EEVS gene; +: m/z features consistent with streptol or streptol-glucoside detected in leaf extracts of host species; n.t.: not tested. Abbreviations: EEVS -2-epi-5-epi-valiolone synthase. All genes of the cluster were found in the same orientation, with the same order. The gene order is preserved in the table, using the of Ca. B. kirkii UZHbot1 accessions as reference.

  . Published chloroplast genomes of Ardisia mamillata (NCBI accession MN136062), Psychotria kirkii (NCBI accession KY378696), Pavetta abyssinica (NCBI accession KY378673), Pavetta schumanniana (NCBI Accession MN851271), and Vangueria infausta (NCBI accession MN851269) were also included in the alignment. Phylogenetic trees were constructed using PhyML v3.3.3 with automatic model selection, and 1000 bootstrap replicates (Guindon et al., 2010). For plant species with uncertain taxonomic identification, seven plant markers were extracted by blastn searches against the metagenome: ITS, nad4, rbcL and rpl16 of Pavetta abyssinica (NCBI accessions MK607930.1, KY492180.1, Z68863.1, and KY378673.1), matK from Pavetta indica (NCBI accession KJ815920.1), petD from Pavetta bidentata (NCBI accession JN054223.1), and trnTF from Pavetta sansibarica (NCBI accession KM592134.1).

Figure 1 :

 1 Figure 1: Phylogeny of Burkholderia, Caballeronia, and Paraburkholderia, including the leaf endophytes. (A) UBCG phylogeny of the Burkholderia s.l. based on 92 conserved genes. Bootstrap support values based on 100 replications are displayed on the branches. Branches with <50% support were collapsed. Ralstonia solanacearum was used as outgroup to root the tree. Coloured samples in boldface represent the leaf endophytes from Rubiaceae and Primulaceae (B) Core genome phylogeny of leaf endophytes based on alignment of 423 singlecopy core genes. Bootstrap support values based on 100 replicates are shown on the branches. Samples are colour-coded based on the host genus: Purple -Ardisia; Blue -Psychotria; Pink -Pavetta; Green -Vangueria; Orange -Fadogia; Black bars represent the coding capacity of the genome (the proportion of the genome coding for functional proteins).

  The most eroded symbionts of Psychotria, Pavetta, and Ardisia lack almost all types of secretion systems, and most also lack a functional flagellar apparatus. Type V secretion systems are present in Ca. Caballeronia ardisicola Acor, Ca. B. pumila UZHbot3 (endophyte of Psychotria pumila), and Ca. B. humilis UZHbot5 (endophyte of Psychotria humilis). The genomes of Paraburkholderia symbionts of Vangueria and Fadogia were generally richer in secretions systems, but only T1SS and T2SS are conserved. A Type V secretion system is present in all Paraburkholderia endophytes except Ca. Paraburkholderia dryadicola Vdry and Vmac (endophytes of V. dryadum and V.

  EEVSs are phylogenetically related to 3-dehydroquinate synthases (DHQS), we aligned the putative EEVS sequences retrieved from leaf endophytes to EEVS and DHQS sequences in the Swissprot database. All putative EEVS sequences retrieved from leaf endophytic Burkholderia were phylogenetically related to bona fide EEVS proteins, but not to dehydroquinate synthase (DHQS) and other sedoheptulose 7-phosphate cyclases. EEVS are otherwise rare in Burkholderia s. l., with putative EEVSs present in only 11 out of 5674 publicly available Burkhoderiaceae genomes (excluding leaf symbiotic bacteria) in the NCBI RefSeq database as of June 2022 (Figure S9).

Figure 2 :

 2 Figure 2: EEVS protein phylogeny and distribution in leaf endophytes. (A) EEVS protein phylogeny of detected EEVS-genes and their closest relatives. Local support values based on the Shimodaira-Hasegawa test are shown on the branches, and branches with support <50% are collapsed. Coloured samples in boldface are the EEVS homologs found in different leaf endophytes. Colours represent different clusters of similar EEVS genes. K-and S-cluster are named after their putative products (K for Kirkamide, and S for Streptol glucoside). NCBI accession numbers of the close relatives are given next to their species name. The tree is rooted using related 3dehydroquinate synthase genes (not shown). *The EEVS gene in Ca. Burkholderia humilis UZHbot5 contains an internal stop codon, creating two EEVS-like pseudogenes. The largest of both was used for the phylogeny. **This EEVS gene of Ca. Burkholderia verschuerenii UZHbot4 is found outside of the K-cluster. Blue and red labels correspond to EEVS sequences belonging to the K and S clusters, respectively. Orange and green labels correspond to EEVS sequences found clustering outside of the K and S clusters, the colour corresponding to the corresponding taxa as in Figure 1. (B) Distribution of specialised metabolism in the leaf endophytes. The phylogenetic tree corresponds to the species phylogeny as in Figure 1A. Samples are colour-coded based on the host species: Purple -Ardisia; Blue -Psychotria; Pink -Pavetta; Green -Vangueria; Orange -Fadogia. Codes next to the species represent presence of specialised metabolite clusters; FR -FR900359 depsipeptide; K -Kirkamide EEVS-cluster; S -Streptol glucoside EEVS-cluster; O -Other EEVS-cluster. K' -Secondary EEVS cluster

  , the common ancestor of Ca. C. ardisicola and Ca. B. crenata possibly possessed both cyclitols and frs pathways, and one of these pathways was lost in the lineages leading to contemporary Ca. B. crenata and Ca. C. ardisicola. Alternatively, the genome of the common ancestor of Ardisia-associated Burkholderia may have encoded cyclitol S-cluster and later acquisition of the frs gene cluster in the Ca. B. crenata lineage alleviated the requirement of EEVS-related metabolism. The model of horizontal acquisition of secondary functions supports the model of endophyte evolution described by Lemaire et al (Lemaire, Vandamme, et al., 2011). Different environmental strains which acquired genes for secondary metabolite production could colonise different host plants in the early open phase of symbiosis. The different phylogenetic endophyte clades observed in the Burkholderia s.l. phylogeny could each represent distinct acquisitions of secondary metabolite gene clusters by divergent free-living bacteria followed by colonisation of different host plants. Many Burkholderia species associate with eukaryotic hosts, including

Table 1 : Genome statistics of newly assembled and re-assembled leaf endophyte genomes.

 1 Coding capacity refers to the proportion of the genome that codes for functional proteins.

	Endophyte	Host species	Type	Assembly	Num. of	N50	GC content	Average	Num. of	Coding	Proportion of	Genome	Genome
				size (Mb)	contigs	(bp)	(%)	coverage	Functional	Capacity (%)	IS	completeness	purity (%)
									genes		elements(%)	(%)
	Ca. Caballeronia ardisicola Acor	Ardisia cornudentata	New assembly	3,95	332	19528	59,23	62x	2026	40,30	0,83	99,28	98,57
	Ca. Burkholderia crenata UZHbot9	Ardisia crenata	Re-assembly	2,65	607	6399	59,02	66x	1670	54,73	2,63	95,68	97,08
	Ca. Caballeronia ardisicola Amam	Ardisia mamillata	New assembly	4,38	333	19687	59,47	13x	2297	40,95	1,10	96,40	97,10
	Ca. Burkholderia crenata Avir	Ardisia virens	New assembly	2,58	605	6517	59,05	13x	1648	56,13	1,64	94,96	99,25
	Paraburkholderia caledonica R-49542	Fadogia homblei	Assembly from isolate	8,92	148	145314	61,59	148x	7695	82,83	1,32	100	100
	Paraburkholderia caledonica R-82532	Fadogia homblei	Assembly from isolate	8,71	123	239289	61,53	168x	7353	81,03	1,05	100	100
	Ca. Caballeronia hochstetteri PhocE	Pavetta hochstetteri	New assembly	3,50	324	18152	62,51	305x	1823	44,53	1,09	99,28	98,57
	Ca. Burkholderia schumanniana UZHbot8	Pavetta schumanniana	Re-assembly	3,62	412	14848	63,47	132x	2453	59,95	1,22	100	97,89
	Ca. Burkholderia brachyanthoides UZHbot7	Psychotria brachyanthoides	Re-assembly	3,75	648	8356	61,00	121x	2109	46,54	3,98	98,56	98,56
	Ca. Burkholderia humilis UZHbot5	Psychotria humilis	Re-assembly	5,32	238	103328	59,60	60x	3264	50,04	1,19	99,28	99,28
	Ca. Burkholderia kirkii UZHbot1	Psychotria kirkii	Reference	4,01	203	44916	62,91	196x *	2069	45,80	8,81	99,28	98,57
	Ca. Burkholderia pumila UZHbot3	Psychotria pumila	Re-assembly	3,70	463	12628	59,13	110x	2192	45,41	4,15	95,68	98,52
	Ca. Burkholderia kirkii UZHbot2	Psychotria punctata	Reference	3,91	48	100248	64,00	-	2539	54,61	9,17	99,28	98,57

X X X X X X X X X X n.t. Ca. Caballeronia ardisicola Amam X X X X X X X X X X n.t. Ca. Caballeronia hochstetteri PhocE X X X Ψ Ψ X X X X X n.t.Ca. Burkholderia humilis UZHbot5X

Acknowledgments

We would like to thank Frédéric De Meyer and Mathijs Deprez for helping with some of the laboratory experiments. We would further like to thank Steven Janssens (Meise Botanic Garden) and Peter Brownless (Royal Botanic Garden Edinburgh) for facilitating the acquisition of plant material for this study. BD and AC would like to thank Klaas Vandepoele, Monica Höfte, Anne Willems and Paul Wilkin for helpful discussion and for proofreading the manuscript. We also thank Aurélien Bailly (University of Zürich, CH) for providing P. kirkii samples and help with interpreting mass spectrometry data. Magda Nel of the H.G.W.J. Schweickerdt Herbarium is thanked for her help with plant identification and Mamoalosi Selepe and Sewes Alberts of the Chemistry and Plant and Soil Sciences Departments, respectively (University of Pretoria) for chemical analysis. We also thank Chien-Chi Hsiao and Karl Gademann from University of Zürich (Switzerland) for providing the analytical standards.

This work was supported by the Flemish Fonds Wetenschappelijk Onderzoek under grant G017717N to AC. AC also acknowledges support from the French National Research Agency under grant agreement ANR-19-TERC-0004-01 and from the French Laboratory of Excellence project "TULIP" (ANR-10-LABX-41; ANR-11-IDEX-0002-02) and from the French National Infrastructure for Metabolomics and Fluxomics, Grant MetaboHUB-ANR-11-INBS-0010. We thank the Oxford Genomics Centre at the Wellcome Centre for Human Genetics for the collection and preliminary analysis of sequencing data. The Oxford Genomics Centre at the Wellcome Centre for Human Genetics is funded by Wellcome Trust grant reference 203141/Z/16/Z. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Notes

The authors declare no conflict of interest.