Le Gac
email: dominique.barloy@agrocampus-ouest.fr

Sequencing, de novo assembly of Ludwigia plastomes, and comparative analysis within the Onagraceae family

Keywords: Water primrose, Ludwigia, Onagraceae, chloroplast genome, long and short reads, hybrid assembly, haplotype

Background

The Onagraceae family, which belongs to the order Myrtales, consists of approximately 657 species and 17 genera, including the genus Ludwigia L., which is comprised of 82 species.

There are few genomic resources for Onagraceae, which limits phylogenetic and population genetics, as well as genomic studies. In this study, new complete plastid genomes of Ludwigia grandiflora subps. hexapetala (Lgh) and Ludwigia peploides subsp montevidensis (Lpm) were generated using a combination of different sequencing technologies. These plastomes were then compared to the published Ludwigia octovalvis (Lo) plastid genome, which was re-annotated by the authors.

Results

We initially sequenced and assembled the chloroplast (cp) genomes of Lpm and Lgh using a hybrid strategy. We observed the existence of two Lgh haplotypes and two potential Lpm haplotypes. Lgh, Lpm, and Lo plastomes were similar in terms of genome size, gene number, structure, and inverted repeat (IR) boundaries, comparable to other species in the Myrtales order. A total of 45 to 65 SSRs (simple sequence repeats), were detected, depending on the species, with the majority consisting solely of A and T, which is common among angiosperms.

Four chloroplast genes (matK, accD, ycf2 and ccsA) were found under positive selection pressure, which is commonly associated with plant development, and especially in aquatic plants such as Lgh, and Lpm.

Conclusion

Our hybrid sequencing approach revealed the presence of two Lgh plastome haplotypes which will help to advance phylogenetic and evolutionary studies, not only specifically for Ludwigia, but also the Onagraceae family and Myrtales order. To enhance the robustness of our findings, a larger dataset of chloroplast genomes would be beneficial.

Introduction

The Onagraceae family belongs to the order Myrtales which includes approximately 657 species of herbs, shrubs, and trees across 17 genera grouped into two subfamilies: subfam. Ludwigioideae W. L. Wagner and Hoch, which only has one genus (Ludwigia L.), and subfam.

Onagroideae which contains six tribes and 21 genera [START_REF] Wagner | Revised classification of the Onagraceae[END_REF]. Ludwigia L. is composed of 83 species [START_REF] Levin | Family-level relationships of Onagraceae based on chloroplast rbcL and ndhF data[END_REF][START_REF] Levin | Paraphyly in tribe Onagreae: insights into phylogenetic relationships of Onagraceae based on nuclear and chloroplast sequence data[END_REF]. The current classification for Ludwigia L., which are composed of several hybrid and/or polyploid species, lists 23 sections. A recent molecular analysis is clarified and supported several major relationships in the genus but has challenged the complex sectional classification of Ludwigia L. [START_REF] Liu | Multi-locus phylogeny of Ludwigia (Onagraceae): insights on infra-generic relationships and the current classification of the genus[END_REF].

The diploid species Ludwigia peploides (Kunth) Raven subsp. montevidensis (Spreng.) Raven (1963) (named here Lpm) (2n=16), and the decaploid species, Ludwigia grandiflora (named here Lgh) (2n=80), reproduce essentially by clonal propagation, which suggests that there is a low genetic diversity within the species (Dandelot et al, 2005). Lgh and Lpm are native to South America and are described as aquatic invasive plants in Europe [START_REF] Hussner | From weed biology to successful control: an example of successful management of Ludwigia grandiflora in Germany[END_REF]. In France, both species occupied aquatic habitats, such as static or slow-flowing waters, riversides, and have recently been observed in wet meadows [START_REF] Lambert | Relationships between the biomass production of invasive Ludwigia species and physical properties of habitats in France[END_REF]. The transition from an aquatic to a terrestrial habitat has led to the emergence of two Lgh morphotypes [START_REF] Haury | Which adaptations of some invasive Ludwigia spp.(Rosidae, Onagraceae) populations occur in contrasting hydrological conditions in Western France?[END_REF]. The appearance of metabolic and morphological adaptations could explain the ability to acclimatize to terrestrial conditions, and this phenotypic plasticity involves various genomic and epigenetic modifications [START_REF] Billet | Aquatic and terrestrial morphotypes of the aquatic invasive plant, Ludwigia grandiflora, show distinct morphological and metabolomic responses[END_REF].

Adequate genomic resources are necessary in order to be able to analyze the different (epi)genomic mechanisms of this acclimation. However, even though the number of terrestrial plant genomes has increased considerably over the last 20 years, only a small fraction (~ 0.16%) have been sequenced, with some clades being significantly more represented than others [START_REF] Marks | Representation and participation across 20 years of plant genome sequencing[END_REF]. Thus, for the Onagraceae family (which includes Ludwigia sp.), only a handful of chloroplast sequences (plastomes) are available, and the complete genome has not yet been sequenced. As of April 2023, there are 10,712 reference plastomes (RefSeq) listed on GenBank, with the vast majority (10,392 genomes) belonging to Viridiplantae (green plants). However, the number of plastomes available for the Onagraceae family is limited, with only 36 plastomes currently listed. Among these, 15 plastomes are from the tribe Epilobieae, with 11 in the Epilobium genus and 4 in the Chamaenerion genus. Additionally, there are 23 plastomes from the tribe Onagreae, with 17 in the Oenothera genus, 5 in the Circaea genus, and only one in the Ludwigia genus.

The Ludwigia octovalvis chloroplast genome was released in 2016 as a unique haplotype of approximately 159 kb [START_REF] Liu | Complete plastome sequence of Ludwigia octovalvis (Onagraceae), a globally distributed wetland plant[END_REF]. L. octovalvis belongs to sect. Macrocarpon (Micheli) H.Hara of the genus Ludwigia while Lpm and Lgh belong to jussieae section [START_REF] Zardini | A new section of Ludwigia (Onagraceae) with a key to the sections of the genus[END_REF], [START_REF] Hoch | The correct name for a section of Ludwigia L.(Onagraceae)[END_REF].

Generally, the inheritance of chloroplast genomes is considered to be maternal in angiosperms.

However, biparentally inherited chloroplast genomes could potentially exist in approximately 20% of angiosperm species [START_REF] Hu | Occurrence of plastids in the sperm cells of Caprifoliaceae: biparental plastid inheritance in angiosperms is unilaterally derived from maternal inheritance[END_REF][START_REF] Zhang | Why does biparental plastid inheritance revive in angiosperms[END_REF] . Both maternal and biparental inheritance are described in the Onagraceae family. In tribe Onagreae, Oenothera subsect. Oenothera are known to have biparental plastid inheritance [START_REF] Wagner | Revised classification of the Onagraceae[END_REF][START_REF] Cleland | Oenothera[END_REF]. In tribe Epilobieae, biparental plastid inheritance was also reported in Epilobium L. with mainly maternal transmission, and very low proportions of paternally transmitted chloroplasts [START_REF] Schmitz | Plastid inheritance in Epilobium[END_REF].

The chloroplast is the symbolic organelle of plants and plays a fundamental role in photosynthesis. Chloroplasts evolved from cyanobacteria through endosymbiosis and thereby inherited components of photosynthesis reactions (photosystems, electron transfer and ATP synthase) and gene expression systems (transcription and translation) [START_REF] Sato | Are Cyanobacteria an Ancestor of Chloroplasts or Just One of the Gene Donors for Plants and Algae?[END_REF]. In general, chloroplast genomes (plastomes) are highly conserved in size, structure, and genetic content.

They are rather small (120-170 kb, [START_REF] Gualberto | The plant mitochondrial genome: dynamics and maintenance[END_REF]), with a quadripartite structure comprising two long identical inverted repeats (IR, 10-30 kb) separated by large and a small single copy regions (LSC and SSC, respectively). They are also rich in genes, with around 100 unique genes encoding key proteins involved in photosynthesis, and a comprehensive set of ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs) [START_REF] Tonti-Filippini | What can we do with 1000 plastid genomes?[END_REF]. Plastomes are generally circular but linear shapes also exist [START_REF] Oldenburg | The linear plastid chromosomes of maize: terminal sequences, structures, and implications for DNA replication[END_REF]. Chloroplast DNA usually represents 5-20% of total DNA extracted from young leaves and therefore low-coverage whole genome sequencing can generate enough data to assemble an entire chloroplast genome [START_REF] Twyford | Strategies for complete plastid genome sequencing[END_REF].

If we refer to their GenBank records, more than 95% of these plastomes were sequenced by so-called short read techniques (mostly Illumina). However, in most seed plants, the plastid genome exhibits two large inverted repeat regions (60 to 335 kb, [START_REF] Twyford | Strategies for complete plastid genome sequencing[END_REF]), which are longer than the short read lengths (< 300 bp). This leads to incomplete or approximate assemblies [START_REF] Wang | Assembly of chloroplast genomes with long-and short-read data: a comparison of approaches using Eucalyptus pauciflora as a test case[END_REF].

Recent long-read sequencing (> 1000 bp) provides strong evidence that terrestrial plant plastomes have two structural haplotypes, present in equal proportions and differing in IR orientation of the [START_REF] Wang | Long-reads reveal that the chloroplast genome exists in two distinct versions in most plants[END_REF]. This shows the importance of using the so-called third generation sequence (TGS, PacBio or Nanopore) to correctly assemble the IRs of chloroplasts and to identify any different structural haplotypes. The current problem with PacBio or Nanopore long read sequencing is the higher error rate compared to short read technology [START_REF] Ferrarini | An evaluation of the PacBio RS platform for sequencing and de novo assembly of a chloroplast genome[END_REF][START_REF] Jain | Nanopore sequencing and assembly of a human genome with ultra-long reads[END_REF][START_REF] Rang | From squiggle to basepair: computational approaches for improving nanopore sequencing read accuracy[END_REF]. Thus, a hybrid strategy which combines long reads (to access the genomic structure) and short reads (to correct sequencing errors) could be effective [START_REF] Wang | Assembly of chloroplast genomes with long-and short-read data: a comparison of approaches using Eucalyptus pauciflora as a test case[END_REF][START_REF] Scheunert | Can we use it? On the utility of de novo and reference-based assembly of Nanopore data for plant plastome sequencing[END_REF].

Here, we report the newly sequenced complete plastid genomes of Ludwigia grandiflora subps. hexapetala (Lgh) and Ludwigia peploides subsp montevidensis (Lpm), using a combination of different sequencing technologies, as well as a re-annotated comparative genomic analysis of the published Ludwigia octovalvis (Lo) plastid. The main objectives of this study are [START_REF] Wagner | Revised classification of the Onagraceae[END_REF] to assemble and annotate the plastomes of two new species of Ludwigia sp., [START_REF] Levin | Family-level relationships of Onagraceae based on chloroplast rbcL and ndhF data[END_REF] to reveal the divergent sequence hotspots of the plastomes in this genus and in the Onagraceae (3) to identify the genes under positive selection.

To achieve this, we utilized long read sequencing data from Oxford Nanopore and short read sequencing data from Illumina to assemble the Lgh plastomes and compared these assemblies with those obtained solely from long reads of Lpm. We also compared both plastomes to the published plastome of Lo. Our findings demonstrated the value of de novo assembly in reducing assembly errors and enabling accurate reconstruction of full heteroplasmy. We also evaluated the performance of a variety of software for sequence assembly and correction in order to define a workflow that will be used in the future to assemble Ludwigia sp. mitochlondrial and nuclear genomes. Finally, the three new Ludwigia plastomes generated by our study make it possible to extend the phylogenetic study of the Onagraceae family and to compare it with previously published analyses [START_REF] Liu | Multi-locus phylogeny of Ludwigia (Onagraceae): insights on infra-generic relationships and the current classification of the genus[END_REF][START_REF] Bedoya | Evolution of the aquatic habit in Ludwigia (Onagraceae): Morpho-anatomical adaptive strategies in the Neotropics[END_REF][START_REF] Liu | Disentangling Reticulate Evolution of North Temperate Haplostemonous Ludwigia (Onagraceae) 1, 2[END_REF].

Material and Methods

Plant sampling and experimental design

The original plant materials were collected in June of 2018 near to Nantes (France) and formal identified by D. Barloy. L. grandiflora subsp. hexapetala (Lgh) plants were taken from the Mazerolles swamps (N47 23.260, W1 28.206), and L. peploides subsp. montevidensis (Lpm) plants from La Musse (N 47.240926, W -1.788688)). Plants were cultivated in a growth chamber in a mixture of 1 /3 soil, 1 /3 sand, 1 /3 loam with flush water level, at 22°C and a 16 h/8 h (light/dark) cycle. A single stem of 10 cm for each species was used for vegetative propagation in order to avoid potential genetic diversity. De novo shoots, taken three centimeters from the apex, were sampled for each species. Samples for gDNA extraction were pooled and immediately snap-frozen in liquid nitrogen, then lyophilized over 48 h using a Cosmos 20K freeze-dryer (Cryotec, Saint-Gély-du-Fesc, France) and stored at room temperature. All the plants were destroyed after being used as required by French authorities for invasive plants (article 3, prefectorial decree n°2018/SEE/2423).

Due to high polysaccharide content and polyphenols in Lpm and Lgh tissues, genomic DNA extraction was carried out using a modified version of the protocol proposed by Panova et al in 2016, with three purification steps [START_REF] Panova | DNA extraction protocols for whole-genome sequencing in marine organisms[END_REF]. 40 mg of lyophilized buds were ground at 30 Hz for 60 s (Retsch MM200 mixer mill, FISHER). The ground tissues were lysed with 1 ml CF lysis buffer (MACHEREY-NAGEL) supplemented with 20 µl RNase and incubated for 1 h at 65°C under agitation. 20 µl proteinase K was then added before another incubation for 1 h at 65°C under agitation. To avoid breaking the DNA during pipetting, the extracted DNA was recovered using a Phase-lock gel tube as described in Belser [START_REF] Belser | Chromosome-scale assemblies of plant genomes using nanopore long reads and optical maps[END_REF]. The extracts were transferred to 2 ml tubes containing phase-lock gel, and an equal volume of PCIA (Phenol, Chloroform, Isoamyl Alcohol; 25:24:1) was added.

After shaking for 5 min, tubes were centrifuged at 11000 g for 20 min. The aqueous phase was transferred into a new tube containing phase-lock gel and extraction with PCIA was repeated.

DNA was then precipitated after addition of an equal volume of binding buffer C4 (MACHEREY-NAGEL) and 99% ethanol overnight at 4°C or 1 h in ice then centrifuged at 800 rpm for 10 min. After removal of the supernatant, 1 ml of CQW buffer was added then the pellet of DNA was re-suspended. Next, DNA purification was carried out by adding a 2 ml mixture of wash buffer PW2 (MACHEREY-NAGEL), wash buffer B5 (MACHEREY-NAGEL), and ethanol at 99% in equal volumes, followed by centrifugation at 800 rpm for 10 min. This DNA purification step was carried out twice. Finally, the DNA pellet was dried in the oven at 70°C for 30 min then re-suspended in 100 µl elution buffer BE (MACHEREY-NAGEL) (5 mM Tris solution, pH 8.5) after 10 min incubation at 65°C under agitation.

A second purification step was performed using a PCR product extraction from gel agarose kit from Macherey-Nagel (MN) NucleoSpin® Gel and PCR Clean-up kit and restarting the above protocol from the step with the addition of CQW buffer then PW2 buffer.

The third purification step consisted of DNA purification using a Macherey-Nagel (MN) NucleoMag kit for clean-up and size selection. Finally, the DNA was resuspended after a 5 min incubation at 65°C in 5 mM TRIS at pH 8.5.

The quantity and quality of the gDNA was verified using a NanoDrop spectrometer, electrophoresis on agarose gel and ethidium bromide staining under UV light and Fragment Analyzer (Agilent Technologies) of the University of Rennes1.

Library preparation and sequencing

MiSeq (Illumina) and GridION (Oxford Nanopore Technologies, referred to here as ONT) sequencing were performed at the PGTB (doi:10.15454/1.5572396583599417E12). Lgh and Lpm genomic DNA were re-purified using homemade SPRI beads (1.8X ratio). For Illumina sequencing, 200 ng of Lgh DNA was used according to the QIAseq FX DNA Library Kit protocol (Qiagen). The final library was checked on TapeStation D5000 screentape (Agilent Technologies) and quantified using a QIAseq Library Quant Assay Kit (Qiagen). The pool was sequenced on an Illumina MiSeq using V3 chemistry and 600 cycles (2x300bp). For ONT sequencing, around 8 µg of Lgh and Lpm DNA were size selected using a Circulomics SRE kit (according to the manufacturer's instructions) before library preparation using a SQK-LSK109 ligation sequencing kit following ONT recommendations. Basecalling in High Accuracy -Guppy version: 4.0.11 (MinKNOW GridION release 20.06.9) was performed for the 48 h of sequencing. Long reads (LR) and short reads (SR) were available for Lgh and only LR for Lpm.

Chloroplast assemblies

Quality controls and preprocessing of sequences were performed using Guppy v4.0.14 base call software for ONT and fastp v0.20.0 [START_REF] Jin | GetOrganelle: a fast and versatile toolkit for accurate de novo assembly of organelle genomes[END_REF] using the default quality filter (phred quality >= Q15) for Illumina reads. After filtering and trimming, Lgh Illumina-reads (SR, 2*23,067,490 reads) were used for reference-guided assembly using GetOrganelle v1.7.0 [START_REF] Jin | GetOrganelle: a fast and versatile toolkit for accurate de novo assembly of organelle genomes[END_REF] and NOVOPlasty v4.2.1 [START_REF] Dierckxsens | NOVOPlasty: de novo assembly of organelle genomes from whole genome data[END_REF] using the parameters recommended for green plants. As we sequenced total DNA, chloroplast reads are mixed with nuclear and mitochondrial reads. Chloroplastic reads were then extracted by mapping against draft Lgh plastomes generated by GetOrganelle in order to reduce computational costs. Chloroplastic R1 and R2 reads were used with and without prior error correction using ONT reads with BayesHammer [START_REF] Nikolenko | BayesHammer: Bayesian clustering for error correction in single-cell sequencing[END_REF] for de novo assemblies using ABySS (version 2.1.5 [START_REF] Jackman | ABySS 2.0: resource-efficient assembly of large genomes using a Bloom filter[END_REF][START_REF] Simpson | ABySS: a parallel assembler for short read sequence data[END_REF]), MEGAHIT (1.1.2, [START_REF] Li | MEGAHIT v1. 0: a fast and scalable metagenome assembler driven by advanced methodologies and community practices[END_REF]), Velvet (version 1.2.10) using VelvetOptimiser and SPAdes (version 3.15.4, [START_REF] Bankevich | SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing[END_REF]). The best k-mer parameters were tested using kmergenie [START_REF] Chikhi | Informed and automated k-mer size selection for genome assembly[END_REF] and Velvetoptimizer. The best results were obtained with k=99, and was therefore the parameter used for all MiSeq assemblers. For ONT reads, Lgh (550,516 reads) and Lpm (68,907 reads) LR reads were self-corrected using CANU 1.8 [START_REF] Koren | Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation[END_REF] and de novo assembly using CANU and FLYE 2.8.2 run with the option --meta and -plasmids [START_REF] Kolmogorov | Assembly of long, error-prone reads using repeat graphs[END_REF]. Hybrid correction and assemblies of LR reads using SR data were realized for Lgh using Ratatosk [START_REF] Holley | Ratatosk: hybrid error correction of long reads enables accurate variant calling and assembly[END_REF].

Post plastome assembly validation

As we used many assemblers and different strategies, we produced multiple contigs that needed to be analyzed and filtered in order to retain only the most robust plastomes. For that, all assemblies were evaluated using the QUality ASsessment Tool (QUAST) for quality assessment (http://cab.cc.spbu.ru/quast/) and visualized using BANDAGE [START_REF] Wick | Bandage: interactive visualization of de novo genome assemblies[END_REF]. BANDAGE compatible graphs (.gfa format) were created with the megahit_toolkit for MEGAHIT and with gfatools for ABySS. Overlaps between fragments were manually checked and ambiguous "IUPAC or N" nucleotides were also biocured with Illumina reads when available.

Chloroplast genome annotation

Plastomes were annotated via the GeSeq [START_REF] Tillich | GeSeq-versatile and accurate annotation of organelle genomes[END_REF] using ARAGORN and tRNAscan_SE to predict tRNAs and rRNAs and tRNAscan_SE to predict tRNAs and rRNAs and via Chloe prediction site (https://chloe.plantenergy.edu.au). The previously reported Lo chloroplast genome was also similarly re-annotated to facilitate genomic comparisons. Gene boundaries, alternative splice isoforms, pseudogenes and gene names and functions were manually checked and biocurated using Geneious (v.10). Finally, plastomes were represented using OrganellarGenomeDRAW (OGDRAW) [START_REF] Greiner | OrganellarGenomeDRAW (OGDRAW) version 1.3. 1: expanded toolkit for the graphical visualization of organellar genomes[END_REF]. These genomes were submitted to GenBank at the National Center of Biotechnology Information (NCBI) with specific accession numbers (for Lgh haplotype 1, (LGH1) OR166254 and Lgh haplotype 2, (LGH2) OR166255; for Lpm haplotype, (LPM) OR166256) using annotation tables generated through GB2sequin [START_REF] Lehwark | GB2sequin-A file converter preparing custom GenBank files for database submission[END_REF].

SSRs and Repeat Sequences Analysis

Simple Sequence Repeats (SSRs) were analyzed through the MISA web server [START_REF] Beier | MISA-web: a web server for microsatellite prediction[END_REF], with parameters set to 10, 5, 4, 3, 3, and 3 for mono-, di-, tri-, tetra-, penta-, and hexa-nucleotides, respectively. Direct, reverse and palindromic repeats were identified using RepEx. Parameters used were: for inverted repeats (min size 15 nt, spacer = local, class = exact); for palindromes (min size 20 nt); for direct repeats (minimum size 30 nt, minimum repeat similarity 97%).

Tandem repeats were identified using Tandem Repeats Finder [START_REF] Benson | Tandem repeats finder: a program to analyze DNA sequences[END_REF], with parameters set to two for the alignment parameter match and seven for mismatches and indels. The IRa region was removed for all these analyses to avoid over representation of the repeats.

Comparative chloroplast genomic analyses

Lgh and Lpm plastomes were compared with the reannotated and biocurated Lo plastome using mVISTA program [START_REF] Frazer | VISTA: computational tools for comparative genomics[END_REF], with the LAGAN alignment algorithm [START_REF] Brudno | LAGAN and Multi-LAGAN: efficient tools for large-scale multiple alignment of genomic DNA[END_REF] and a cut-off of 70% identity.

Nucleotide diversity (Pi) was analyzed using the software DnaSP v.6.12.01 [START_REF] Rozas | DnaSP 6: DNA sequence polymorphism analysis of large data sets[END_REF][START_REF] Rozas | DnaSP version 3: an integrated program for molecular population genetics and molecular evolution analysis[END_REF] with step size set to 200 bp and window length to 300 bp. IRscope [START_REF] Amiryousefi | IRscope: an online program to visualize the junction sites of chloroplast genomes[END_REF] was used for the analyses of inverted repeat (IR) region contraction and expansion at the junctions of chloroplast genomes.

To assess the impact of environmental pressures on the evolution of these three Ludwigia species, we calculated the nonsynonymous (Ka) and synonymous (Ks) substitutions and their ratios (ω = Ks/Ks) using TBtools [START_REF] Chen | TBtools: an integrative toolkit developed for interactive analyses of big biological data[END_REF] to measure the selective pressure. Genes with ω < 1, ω = 1, and 1 < ω were considered to be under purifying selection (negative selection), neutral selection, and positive selection, respectively.

Plastome phylogenomic analyses

We reconstructed phylogenetic relationships among plastomes of Onagraceae. The FFT-NS-2 method in MAFFT 7 [START_REF] Katoh | MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform[END_REF] was used to align all plastomes with one of the IRs removed to avoid data duplication. Phylogenetic tree analysis, based on the maximum likelihood (ML) method, was conducted in RAxML 8.2.9 [START_REF] Stamatakis | The RAxML v8. 2. X Manual[END_REF] using the GTR+G model with node support assessed by fast-bootstrap (-f a) using 1,000 non-parametric bootstrap pseudo-replicates.

Graphic representation

Statistical analyses were performed using R software in RStudio integrated development environment (R Core Team, 2015, RStudio: Integrated Development for R. RStudio, Inc., Boston, MA, http://www.rstudio.com/). Figures were realized using ggplot2, ggpubr, tidyverse, dplyr, gridExtra, reshape2, and viridis packages. SNPs were represented using trackViewer [START_REF] Amiryousefi | IRscope: an online program to visualize the junction sites of chloroplast genomes[END_REF] and genes represented using gggenes packages.

Results

Plastome short read assembly

We first used GetOrganelle and Novoplasty with the short reads of Lgh. GetOrganelle reconstructed two identical haplotypes that differ only by a "flip-flop" of the SSC region, as shown in Figure 1, while NOVOplasty generated three contigs that can be reassembled to form either one of the two haplotypes, as the SSC region can be oriented in both directions.

In order to evaluate the best strategies for assembly of Lgh plastomes, we used these haplotypes to map and extract short (Illumina) and long (Nanopore) chloroplast reads.

Chloroplastic short reads, with or without prior correction using long reads, were assembled using ABySS, Velvet, MEGAHIT and SPAdes. As shown in Figure 2A, the number of contigs obtained is highly variable depending on the tools and strategies. Without correction, Velvet and MEGAHIT generate highly fragmented assemblies with 1103 and 722 contigs respectively, while ABySS and SPAdes produce a more reasonable number of contigs (65 and 12 respectively). In addition, all contigs produced by all assemblers except SPAdes, are predominantly less than 500 nt long (Figure 2B from 68 to 95% of the contigs). By correcting the reads before assembly, we saw a noticeable improvement in the number of contigs, which decreased for all assemblers while increasing their sizes. However, except for SPAdes, the number of contigs below 500 nt remained preponderant (from 54 to 70%, Figure 2C).

An assessment of the assembly quality using QUAST, and based on an alignment on Lgh haplotype 1 generated by GetOrganelle as a reference, shows that none of the assemblers produces a complete plastome. By comparing the contribution of small contigs (less than 200, 500 and 1000 nt), we noticed an invariant coverage rate for SPAdes (85%), while the fraction of covered genome decreases drastically for Velvet (from 92% to 22%), correlated with a duplication rate going from 2 to 1 (Add. Figure 1A). We noted that compared to an assembly with all reads, the use of reads superior to 1 kb slightly decreases the coverage rate to reach an optimal duplication ratio equal to 1. The effect of prior read correction is notable for MEGAHIT and Velvet, especially concerning the increase in the size of the large alignment (Add. Figure 1A), loss of misassemblies, and reduction of the number of mismatches (Add. Figure 1B). Investigating all possible scaffolds generated by BANDAGE visualization (Add. Figure 2)

shows that ABySS and SPAdes assembly graphs suggest a tripartite structure with the long single-copy (LSC) region as the larger circle in the graph (blue), joined to the small single-copy region (green) by one copy of the inverted repeats (IRs, red). In all graphs, the two IRs are a collapsed repeat of approximately twice the coverage. For Velvet and MEGAHIT, graphs confirm the significant fragmentation of the assemblies, which is improved by prior correction of the reads.

In conclusion, SPAdes is the most efficient tool, and gives the best result using corrected short reads to assemble a resolved plastome, with three contigs of 90,272 bp (corresponding to LSC), [START_REF] Oldenburg | The linear plastid chromosomes of maize: terminal sequences, structures, and implications for DNA replication[END_REF],788 bp (corresponding to SSC) and 24,762 bp (corresponding to a copy of the IR).

Plastome long read assembly

Chloroplast fractions of Lgh long reads were selected through mapping. Raw, selfcorrected reads were assembled using CANU, and short-read-corrected comparative assembly was carried out using CANU or FLYE. Using raw data, CANU generates a unique contig corresponding to haplotype 2, whereas FLYE makes two contigs that reconstruct haplotype 1.

Assemblies using long reads corrected by CANU leads to fragmentation into two (CANU assembler) or three (FLYE assembler) contigs which both reconstruct haplotype 1, with an large gap corresponding to one of the IR copies for the CANU assembler. Finally, correction by RATATOSK allows CANU to assemble two redundant contigs, with contig 1 reproducing the entire haplotype 2, while FLYE makes two contigs corresponding to haplotype 1 (Add. Figure 3A). In conclusion, the two haplotypes proposed by GetOrganelle using the short reads were likewise reconstructed randomly using the long reads, with or without correction.

Consequently, both haplotypes were retained as Lgh plastomes and submitted to Genbank.

Hybrid assembly is the most complete and the most faithful, with an average of 1.6 contigs (all assemblers combined), and optimal correction using RATATOSK, which permits plastomes with 99.94% accuracy compared to biocurated molecules (Add. Figure 3B).

Unfortunately, due to the absence of short read data, we could only perform self-corrected long read assembly for Lpm using CANU. We also compared CANU and FLYE assembler efficiency, and found that assembly using CANU produces 13 contigs whereas FLYE produces 12 contigs. In both cases, only three contigs are required to reconstitute a complete cpDNA assembly (no gap, no N), with an SSC region oriented like those of the Lgh haplotype 2 and the Lo plastome. Although it is more than likely that these two SSC region orientations also exist for Lpm, the low number of nanopore sequences generated (68907 reads) and absence of Illumina short reads prevented us from demonstrating the existence of both haplotypes. As a result, only the "haplotype 2" generated sequence was deposited to Genbank.

Annotation and comparison of Ludwigia plastomes 1. General Variations

Plastomes of the three species of Ludwigia sp., Lgh, Lpm and Lo, are circular doublestranded DNA molecules (Figure 3) which are all (as shown in Table 1) approximately the same size: Lo being slightly smallest and Lgh the biggest. The overall GC content is almost the same for the three species and the GC contents of the IR regions are higher than those of the LSC and SSC regions. Between the three species, the lengths of the total chloroplasts, LSC, SSC, and IR are broadly similar (Table 2) and the three plastomes are perfectly syntenic if we orient the SSC fragments the same way.

All three Ludwigia sp. plastomes contain the same number of functional genes (134 in total) encoding 85 proteins (embracing 7 duplicated in the IR region: ndhB, rpl2, rpl23, rps7, rps12, ycf2, ycf15), 37 tRNAs (including trnK-UUU which contains matK), and 8 rRNAs (16S, 23S, 5S, and 4.5S as duplicated sets in the IR). Among these genes, 18 contain introns, of which six are tRNAs (Table 2). Only the rps12 gene is a trans-spliced gene. A total of 46 genes are involved in photosynthesis, and 71 genes related to transcription and translation, including a bacterial-like RNA polymerase and 70S ribosome, as well as a full set of transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs). Six other protein-coding genes are involved in essential functions, such as accD, which encodes the β-carboxyl transferase subunit of acetyl-CoA carboxylase, an important enzyme for fatty acid synthesis; matK encodes for maturase K, which is involved in the splicing of group II introns; cemA, a protein located in the membrane envelope of the chloroplast is involved in the extrusion of protons and therby indirectly allows the absorption of inorganic CO2 in the plastids; clpP1 which is involved in proteolysis, and; ycf1, ycf2, two ATPases members of the TIC translocon. Finally, a highly pseudogenized ycf15 locus was annotated in the IR even though premature stop codons indicate loss of functionality.

Segments Contractions/Expansion

The junctions between the different chloroplast segments were compared between three Ludwigia sp. (Lpm, Lgh and Lo), and we found that the overall resemblance of Ludwigia sp.

plastomes was confirmed at all junctions (Figure 4A). In all three genomes, rpl22, rps19, and rpl2 were located around the LSC/IRb border, and rpl2, trnH, and psbA were located at the IRa/LSC edge. The JSB (junction between IRb and SSC) is either located in the ndhF gene or the ycf1 gene depending on the orientation of the SSC region (Figure 4B). The ycf1 gene was initially annotated as a 1139 nt pseudogene that we biocurate as a larger gene (5302 nt) with a frameshift due to a base deletion, compared to Lg and Lo which both carry a complete ycf1 gene.

If we compare Ludwigia sp. junctions with those of other Onagraceae plastomes (Figure 5), we can observe that the JLB and JLA connections are well-preserved in the whole family, whereas JSB and JLA differ. Concerning JSB, in the five Onagraceae family genera with available plastomes, ndhF is duplicated, with the exception of Circaea sp. and Ludwigia sp. For Oenothera villosa, the first copy of ndhF, which is located in the IRb, overlaps the JSB border, whereas for Oenothera lindheimeri, Epibolium amurense and Chamaenerion sp., ndhF is only located in inverted repeats. Only Circaea sp. and Ludwigia sp. have a unique copy of this locus, and it is found in the SSC segment (Figure 5). At the JSA border, in Circaea sp., the ycf1 gene crosses the IRa/SSC boundary and extends into the IRa region.

When comparing the respective sizes of chloroplast fragments (IR/SSC/LSC) in Onagraceae, it can be observed that Ludwigia species exhibit expansions in the SSC and LSC regions which are not compensated by significant contractions in the IR regions. This is likely due to the relocation of the ndhF in the SSC region and rps19 in the LSC region. Additionally, there may be significant size variations in the intergenic region between trnI and ycf2, as well as the intergenic segment containing the ycf15 pseudogene (Add. Figure 4).

Repeats and SSRs analysis

In this study, we analyzed the nature and distribution of single sequence repeats (SSR), as their polymorphism is an interesting indicator in phylogenetic analyses. A total of 65 (Lgh), 48 (Lpm) and 45 (Lo) SSRs were detected, the majority being single nucleotide repeats , followed by tetranucleotides (12-10) and then di-, tri-and penta-nucleotides (Add. Figure 5A).

Mononucleotide SSRs are exclusively composed of A and T, indicating a bias towards the use of the A/T bases, which is confirmed for all SSRs (Add. Figure 5B). In addition, the SSRs are mainly distributed in the LSC region for the three species, which is probably biased by the fact that LSC is the longest segment of the plastome (Add. Figure 5C). The analysis of SRR locations revealed that most were distributed in non-coding regions (intergenic regions and introns, Add. Figure 5D).

The chloroplast genomes of the three Ludwigia species were also screened for long repeat sequences. They were counted in a non-redundant way (if smaller repetitions were included in large repeats, only the large ones were considered). Four types of repeats (tandem, palindromic inverted and direct) were surveyed in the three Ludwigia sp. plastomes. No inverted repeats were detected with the criteria used.

For the three other types of repeats, here are their distributions:

Tandem repeats (Table 3A): Perfect tandem repeats (TRs) with more than 15 bp were examined. Twenty-two loci were identified in the three Ludwigia sp. plastomes (Lgh, Lpm, Lo), heterogeneously distributed as shown in Table 3A: 13 loci (plus one imperfect) in Lo, nine loci (plus one imperfect) in Lgh and seven loci (plus two imperfect) in Lpm. It can therefore be seen that the TR distributions (occurrence and location) are specific to each plastome, since only four pairs are common to the three species. Thus, nine TRs are unique to Lo, three to Lpm and three to Lgh. Two pairs are common to Lgh and Lpm and one is common to Lo and Lgh. TRs are mainly intergenic or intronic but are detected in two genes (accD and ycf1). These genes have accelerated substitution rates, although this does not generate a large difference in their lengths. This point will be developed later in this article. 3B): There are few direct (non-tandem) repeats (DRs) in the chloroplast genomes of Ludwigia sp. A single direct repeat of 41 nt is common to the three species, at 2 kb intervals, in psaB and psaA genes. This DR corresponds to an amino acid repeat 3C): Palindromic repeats make up the majority of long repetitions, with the numbers of perfect repeats varying from 19, 24 and 26 in Lo, Lgh and Lpm, respectively, and the number of quasi-palindromes (1 mutation) varying between 8, 3 and 6.

Direct repeats (Table

Palindromes (Table

They are mainly found in the intronic and intergenic regions, with the exception of six genic locations in psbD, ndhK, ccsA and rpl22, and two palindromic sequences in ycf2. These gene palindromic repeats do not seem to cause genetic polymorphism in Ludwigia and can be considered as silent.

Thirteen palindromes are common to the three species (including 2 with co-variations in Lo). 13 others present in Lpm and Lgh correspond to quasi-palindromes (QPs) in Lo due to mutated bases, and conversely, three Lo perfect palidromes are mutated in Lpm and Lgh.

Finally, only five palindromes are species specific. Two in particular are located in the hypervariable intergenic spacer ndhF-rpl32, and are absent in Lo due to a large deletion of 160 nt.

Repeat distribution in LSC, SSC and IR segments

In the IRa/IRb regions, repeats are only identified in the first 9 kb region between rpl2 and ycf2: a tandem repeat in the Lpm rpl2 intron, and a tetranucleotide repeat, [TATC]*3, located in the ycf2 gene in the 3 species. In ycf2 we also found 1 common palindrome (16 nt), a single palindrome in Lo (20 nt, absent following an A:G mutation in the 2 other species), as well as a shared tandem repeat (24 nt), and an additional 15 nt tandem repeat in Lo which adds 4 amino acids to protein sequence.

In the SSC region, the repeats are almost all located in the intergenic and/or intronic regions, with a hotspot between ndhF and ccsA. There is also a shared microsatellite in ndhF, and a palidrome (16 nt) in ccsA which is absent in Lo (due to an A:C mutation), resulting in a synonymous mutation (from isoleucine to leucine). We also observed multiple and various repeats in the ycf1 gene: 3 common poly-A repeats (from 10 to 13 nt), 3 species-specific microsatellites (ATAG)*3 and (ACCA)*4 in Lgh and (CAAC)*3 in Lo, as well as two direct repeats of 32 nt (37 nt spacing), which were absent from Lo due to a G:T SNP. Two tandem repeats were also observed in Lo and Lgh. Neither of these repeats are at the origin of the frameshift causing the pseudogenization of ycf1 in Lo, this latter being due to a single deletion of an A at position 3444 of the gene.

Finally, in the LSC region, the longest segment, which consequently contains the maximum number of repeats, we still observed a preferential localization in the intergenic and intronic regions since only genes atpA, rpoC2, rpoB, psbD, psbA, psbB, ndhK and clpP1 contain either mononucleotic repeats (poly A and T), palindromes, or microsatellites (most often common to the three species and without affecting the sequences of the proteins produced). As mentioned earlier, the only exception is the accD gene, which contains several direct and tandem repeats in Lgh and Lpm, corresponding to a region of 174 nt (58 amino acids) missing in Lo and, conversely, a direct repeat of 40 nucleotides, in a region of 147 nt (49 aa), which is present in Lo and missing in the other two species. These tandem repeats lead to the presence of four Figure 6). It should be noted that though these TRs do not disturb the open reading phases, it is still possible for them to form an intron which is not translated. Different functional studies will be necessary to clarify this point. The presence of polymorphisms of the accD gene between Lo and the two species (Lpm, Lgh) is interesting because accD, that encodes a subunit of acetyl-CoA carboxylase (EC 6.4.1.2). This enzyme is essential in fatty acid synthesis and also catalyzes the synthesis of malonyl-CoA, which is necessary for the growth of dicots, plant fitness and leaf longevity, and is involved in the adaptation to specific ecological niches [START_REF] Konishi | Compartmentalization of two forms of acetyl-CoA carboxylase in plants and the origin of their tolerance toward herbicides[END_REF].

Large accD expansions due to TRs have also been described in other plants such as Medicago [START_REF] Wu | Extensive genomic rearrangements mediated by repetitive sequences in plastomes of Medicago and its relatives[END_REF] and Cupressophytes [START_REF] Li | The Repeat Sequences and Elevated Substitution Rates of the Chloroplast accD Gene in Cupressophytes[END_REF]. Some authors have suggested that these inserted repeats are not important for acetyl-CoA carboxylase activity as the reading frame is always preserved, and they assume that these repeats must have a regulatory role [START_REF] Gurdon | Two distinct plastid genome configurations and unprecedented intraspecies length variation in the accD coding region in Medicago truncatula[END_REF].

Sequence Divergence Analysis and Polymorphic Loci Identification

Determination of divergent regions by MVista, using Lo as a reference, confirmed that the three Ludwigia sp. plastomes are well preserved if the SSC segment is oriented in the same way (Add. Figure 7). Sliding window analysis (Figure 6) indicated variations in definite coding regions, notably clpP, accD, ndh5, ycf1 with high Pi values, and to a lesser extent, rps16, matK, ndhK, petA, ccsA and four tRNAs (trnH,trnD, trnT and trnN). These polymorphic loci could be suitable for inferring genetic diversities in Ludwigia sp.

A comparative analysis of the sizes of protein coding genes sizes also shows that the rps11 gene initially annotated in Lo is shorter than those which have been newly annotated in Lgh and Lpm (345 bp instead of 417 bp). Comparative analysis by BLAST shows that it is the long form which is annotated in other Myrtales, and the observation of the locus in Lo shows a frameshift mutation (deletion of a nucleotide in position 311). Functional analysis would be necessary to check whether the rps11 frameshift mutation produces shorter proteins that have lost their function. And only obtaining the complete genome will verify whether copies of some of these genes have been transferred to mitochondrial or nuclear genomes. Such rps11 horizontal transfers have been reported for this gene in the mitochondrial genomes of various plant families [START_REF] Richardson | Horizontal gene transfer in plants[END_REF]. This also applies to ycf1, found as a pseudogene in Lo (as specified previously), although it is not known if this reflects a gene transfer or a complete loss of function [START_REF] De Vries | YCF1: A Green TIC?[END_REF][START_REF] Filip | Horizontal Gene Transfer Involving Chloroplasts[END_REF].Moreover, there is a deletion of nine nucleotides in the 3' region of the rpl32 gene in Lgh and Lpm, leading to a premature end of the translation and the deletion of the last 4 amino acids [QRLD], which are replaced by a K. However, if we look carefully at the preserved region as defined by the RPL32 domain (CHL00152, member of the superfamily CL09115), we see that the later amino acids are not important for rpl32 function since they are not found in the orthologs.

Our results show that the Ka/Ks ratio is less than 1 for most genes (Figure 7). This indicates adaptive pressures to maintain the protein sequence except for matK (1.17 between Lgh and Lpm), accD (2.48 between Lgh and Lo and 2.16 between Lpm and Lo), ycf2 (4.3 between both Lgh-Lp and Lo) and ccsA (1.4 between both Lgh-Lpm and Lo), showing a positive selection for these genes, and a possible key role in the processes of the species' ecological adaptations. As we have already described the variability in the accD sequence, we will focus on ycf2, matK, and ccsA variations.

Concerning ccsA, the variations observed, although significant, concern only five amino acids, and modifications do not seem to affect the C-type cytochrome synthase gene function.

Concerning ycf2, our analysis shows that this gene is highly polymorphic with 256 SNPs that provoke 10 deletions, 7 insertions, 21 conservative and 49 non-conservative substitutions in Lo (Add. Figure 8), compared to Lgh and Lpm (100 % identical). This gene has been shown as "variant" in other plant species such as Helianthus tuberosus [START_REF] Zhong | The complete chloroplast genome of the Jerusalem artichoke (Helianthus tuberosus L.) and an adaptive evolutionary analysis of the ycf2 gene[END_REF].

The matK gene has been used as a universal barcoding locus to enable species discrimination of terrestrial plants [START_REF] Antil | DNA barcoding, an effective tool for species identification: a review[END_REF], and is often, together with the rbcL gene, the only known genetic resource for many plants. Thus, we propose a phylogenetic tree from Ludwigia matK sequences (Figure 8). It should however be noted that this tree contains only 149 amino acids common to all the sequences (out of the 499 in the complete protein). As only three complete Ludwigia plastomes are available, we cannot specify whether these barcodes are faithful to the phylogenomic history of Ludwigia in the same way as the complete plastome. In any case, for this tree, we can see that Lo stands apart from the other Ludwigia sp., Lpm and Lgh, and that the L. grandiflora subsp. hexapetala belongs to the same branch as the species L. ovalis (aquatic taxon used in aquariums [START_REF] Li | Removal effects of aquatic plants on high-concentration phosphorus in wastewater during summer[END_REF]), L. stolonifera (native to the Nile, found in a variety of habitats, from freshwater wetlands to brackish and marine waters) [START_REF] Soliman | Ludwigia stolonifera (Guill. & Perr.) PH Raven, insight into its phenotypic plasticity, habitat diversity and associated species[END_REF] and L. adscendens (common weed of rice fields in Asia) [START_REF] Kamoshita | Residual effects of cultivation methods on weed seed banks and weeds in Cambodia[END_REF]. Lpm is in a branch unique to its species but close to the L. grandiflora subsp. hexapetala branch.

Discussion

In the present study, we first sequenced and de novo assembled the chloroplast (cp) genomes of Ludwigia peploides (Lpm) and Ludwigia grandiflora (Lgh), two species belonging to the Onagraceae family. We employed a hybrid strategy and demonstrated the presence of two cp haplotypes in Lgh and one haplotype in Lpm, although the presence of both haplotypes in Lpm is likely. Furthermore, we compared these genomes with those of other species in the Onagraceae family to expand our knowledge of genome organization and molecular evolution in these species.

Our findings demonstrate that the utilization of solely short reads has failed to produce complete plastomes, likely due to challenges posed by long repeats and rearrangements. On the other hand, relying solely on long reads resulted in a lower quality sequence due to insufficient coverage and sequencing errors. After conducting our research, we discovered that hybrid assembly, which incorporates both long and short read sequences, resulted in the most superior complete assemblies. This innovative approach capitalizes on the advantages of both sequencing technologies, harnessing the accuracy of short read sequences and the length of long read sequences. Our findings corroborate with similar results obtained in studies on other chloroplasts, such as those in Eucalyptus [START_REF] Wang | Assembly of chloroplast genomes with long-and short-read data: a comparison of approaches using Eucalyptus pauciflora as a test case[END_REF], Falcataria [START_REF] Anita | Chloroplast genome draft assembly of Falcataria moluccana using hybrid sequencing technology[END_REF], Carex [START_REF] Xu | Chloroplast genomes of four Carex species: Long repetitive sequences trigger dramatic changes in chloroplast genome structure[END_REF] or Cypripedium [START_REF] Guo | Chloroplast Genomes of Two Species of Cypripedium: Expanded Genome Size and Proliferation of AT-Biased Repeat Sequences[END_REF].

As a result of this strategy, we were able to successfully identify the presence of two haplotypes in Lgh, which is a first for Ludwigia, as the plastome of L. octovalvis was only delivered in the form of one haplotype [START_REF] Liu | Complete plastome sequence of Ludwigia octovalvis (Onagraceae), a globally distributed wetland plant[END_REF]. Due to the unavailability of sequence data for Ludwigia octovalvis and our exclusive use of long reads for Ludwigia peploides, we are unable to conclusively identify the presence of these two forms in the Ludwigia genus. However, we believe that they are likely to be present. Unfortunately, the current representation of plastomes in GenBank primarily consists of short-read data, which may result in an underrepresentation of this polymorphism. It is unfortunate that structural heteroplasmy, which is expected to be widespread in angiosperms, has been overlooked. Notably, the existence of two plastome haplotypes has been widely identified in the closely related order of Myrtales (Eucalyptus sp.), and more commonly in Angiosperms, with 58 species across 16 other orders showing similar patterns [START_REF] Wang | Long-reads reveal that the chloroplast genome exists in two distinct versions in most plants[END_REF]. Recent studies have also revealed the presence of two cp haplotypes in other orders, such as Asparagales (Ophrys apifera orchid, [START_REF] Bateman | Whole plastomes are not enough: phylogenomic and morphometric exploration at multiple demographic levels of the bee orchid clade Ophrys sect. Sphegodes[END_REF]), Brassicales (Carica papaya, Vasconcellea pubescens, [START_REF] Lin | Comparative analysis of chloroplast genomes in Vasconcellea pubescens A.DC. and Carica papaya[END_REF]), Solanales (Solanum tuberosum, [START_REF] Lihodeevskiy | The Use of Long-Read Sequencing to Study the Phylogenetic Diversity of the Potato Varieties Plastome of the Ural Selection[END_REF]), and even in a new order, Laurales (Avocado Persea americana, [START_REF] Nath | A haplotype resolved chromosomal level avocado genome allows analysis of novel avocado genes[END_REF]). Furthermore, [START_REF] Wanichthanarak | Revisiting chloroplast genomic landscape and annotation towards comparative chloroplast genomes of Rhamnaceae[END_REF] revisited 24 Ramanaceae published chloroplast genomes and identified a likely form of heteroplasmy in Rhamnus crenata [START_REF] Wanichthanarak | Revisiting chloroplast genomic landscape and annotation towards comparative chloroplast genomes of Rhamnaceae[END_REF]. These emerging findings highlight the potential underestimation of the occurrence of two chloroplast haplotypes in angiosperms and emphasize the importance of identifying and considering such heteroplasmy during de novo or revisited chloroplast genome assemblies.

The chloroplast genome sizes for the three genera of Onagraceae subfam. Onagroideae varied as follows: Circaea sp. ranged from 155,817 bp to 156,024 bp, Chamaenerion sp. ranged from 159,496 bp to 160,416 bp, and Epilobium sp. ranged from 160,748 bp to 161,144 bp [START_REF] Luo | Comparative Analysis of Complete Chloroplast Genomes of 13 Species in Epilobium, Circaea, and Chamaenerion and Insights Into Phylogenetic Relationships of Onagraceae[END_REF]. Our study revealed that the size of the complete chloroplast of Ludwigia (Onagraceae subfamily Ludwigioideae) ranged from 159,369 bp to 159,584 bp, which is remarkably similar to other Onagraceae plants (average length of 162,030 bp). Furthermore, Ludwigia plastome sizes are consistent with the range observed in Myrtales (between 152,214 to 171,315 bp, [START_REF] Zhang | Comparative analysis of chloroplast genome structure and molecular dating in Myrtales[END_REF]). In the same way, similar overall GC content was found in Ludwigia sp. (from 37.3 to 37.4%), Circaea sp. (37.7 to 37.8%), Chamaenerion sp. and Epilobium sp. (38.1 to 38.2%, [START_REF] Luo | Comparative Analysis of Complete Chloroplast Genomes of 13 Species in Epilobium, Circaea, and Chamaenerion and Insights Into Phylogenetic Relationships of Onagraceae[END_REF]) and more generally for the order Myrtales (36.9-38.9%, with the average GC content being 37%, [START_REF] Zhang | Comparative analysis of chloroplast genome structure and molecular dating in Myrtales[END_REF]).

Higher GC content of the IR regions (43.5%) found in Ludwigia sp. has already been shown in the Myrtales order (39.7-43.5%) and in other families/orders such as Amaranthaceae (order Caryophyllales, [START_REF] Xu | Comparing and phylogenetic analysis chloroplast genome of three Achyranthes species[END_REF]) or Lamiaceae (order Lamiales, [START_REF] Lian | Comparative analysis of chloroplast genomes reveals phylogenetic relationships and intraspecific variation in the medicinal plant Isodon rubescens[END_REF]), and is mainly due to the presence of the four GC rich rRNA genes.

The complete chloroplast genomes of the three Ludwigia species encoded an identical set of 134 genes including 85 protein-coding genes, 37 tRNA genes and eight ribosomal RNAs, consistent with gene content found in the Myrtales order, with a gene number varying from 123 to 133 genes with 77-81 protein-coding genes, 29-31 tRNA gene and four rRNA genes [START_REF] Zhang | Comparative analysis of chloroplast genome structure and molecular dating in Myrtales[END_REF].

Chloroplast genes have been selected during evolution due to their functional importance [START_REF] Mohanta | Gene Loss and Evolution of the Plastome[END_REF].

In our current study, we made the noteworthy discovery that matK, accD, ycf2, and ccsA genes were subjected to positive selection pressure. These genes have frequently been reported in literature as being associated with positive selection, and are known to play crucial roles in plant development conditions. Lgh and Lpm are known to thrive in aquatic environments, where they grow alongside rooted emergent aquatic plants, with their leaves and stems partially submerged during growth, as reported by Wagner et al. in 2007 [START_REF] Wagner | Revised classification of the Onagraceae[END_REF]. Both species possess the unique ability of vegetative reproduction, enabling them to establish themselves rapidly in diverse habitats, including terrestrial habitats, as noted by Haury et al [START_REF] Haury | Which adaptations of some invasive Ludwigia spp.(Rosidae, Onagraceae) populations occur in contrasting hydrological conditions in Western France?[END_REF]. Additionally, Lo is a wetland plant that typically grows in gullies and at the edges of ponds, as documented by Wagner et al. in 2007 [1]. Given their ability to adapt to different habitats, these species may have evolved specialized mechanisms to cope with various abiotic stresses, such as reduced carbon and oxygen availability or limited access to light in submerged or emergent conditions.

Concerning matK, Barthet et al [START_REF] Barthet | Expression of matK: functional and evolutionary implications[END_REF] demonstrated the relationship between light and developmental stages, and MatK maturase activity, suggesting important functions in plant physiology. This gene has recently been largely reported to be under positive selection in an aquatic plant (Anubias sp., [START_REF] Li | Comparative Analyses of Plastomes of Four Anubias (Araceae) Taxa, Tropical Aquatic Plants Endemic to Africa[END_REF]), and more generally in terrestrial plants (Pinus sp, [START_REF] Zeb | Comparative genome sequence and phylogenetic analysis of chloroplast for evolutionary relationship among Pinus species[END_REF] or Chrysosplenium sp. [START_REF] Wu | Analysis of six chloroplast genomes provides insight into the evolution of Chrysosplenium (Saxifragaceae)[END_REF]). The accD gene has been described as an essential gene required for leaf development [START_REF] Kode | The tobacco plastid accD gene is essential and is required for leaf development[END_REF] and longevity [START_REF] Madoka | Chloroplast transformation with modified accD operon increases acetyl-CoA carboxylase and causes extension of leaf longevity and increase in seed yield in tobacco[END_REF] in tobacco (Nicotiana tabacum). Under drought stress, plant resistance can be increased by inhibiting accD [START_REF] Gu | Drought stress triggers proteomic changes involving lignin, flavonoids and fatty acids in tea plants[END_REF], and conversely, enhanced in response to flooding stress by upregulating accD accumulation [START_REF] Bharadwaj | Physiological and genetic responses of lentil (Lens culinaris) under flood stress[END_REF]. Hence, we can hypothesize that the positive selection observed on the accD gene can be explained by the submerged and emerged constraints undergone by Ludwigia species. The ycf2 gene seems to be subject to adaptive evolution in Ludwigia species. Its function, although still vague, would be to contribute to a protein complex generating ATP for the TIC machinery (proteins importing into the chloroplasts [START_REF] Kikuchi | A Ycf2-FtsHi Heteromeric AAA-ATPase Complex Is Required for Chloroplast Protein Import[END_REF][START_REF] Schreier | Plastidial NAD-Dependent Malate Dehydrogenase: A Moonlighting Protein Involved in Early Chloroplast Development through Its Interaction with an FtsH12-FtsHi Protease Complex[END_REF]), as well as plant cell survival [START_REF] Drescher | The two largest chloroplast genome-encoded open reading frames of higher plants are essential genes[END_REF][START_REF] Xing | The plastid-encoded protein Orf2971 is required for protein translocation and chloroplast quality control[END_REF]. The ccsA gene positive selection is found in some aquatic plants such as Anubia sp. [START_REF] Li | Comparative Analyses of Plastomes of Four Anubias (Araceae) Taxa, Tropical Aquatic Plants Endemic to Africa[END_REF], marine flowering plants as Zostera species [START_REF] Chen | Chloroplast genomic comparison provides insights into the evolution of seagrasses[END_REF], and some species of Lythraceae [START_REF] Gu | Drought stress triggers proteomic changes involving lignin, flavonoids and fatty acids in tea plants[END_REF]. The ccsA gene is required for cytochrome c biogenesis [START_REF] Xie | The plastid-encoded ccsA gene is required for heme attachment to chloroplast c-type cytochromes[END_REF] and this hemoprotein plays a key role in aerobic and anaerobic respiration, as well as photosynthesis [START_REF] Kranz | Molecular mechanisms of cytochrome c biogenesis: three distinct systems[END_REF]. Furthermore, we showed that Lgh colonization is supported by metabolic adjustments mobilizing glycolysis and fermentation pathways in terrestrial habitats, and the aminoacyl-tRNA biosynthesis pathway, which are key components of protein synthesis in aquatic habitats [START_REF] Billet | Aquatic and terrestrial morphotypes of the aquatic invasive plant, Ludwigia grandiflora, show distinct morphological and metabolomic responses[END_REF] It can be assumed that the ability of Ludwigia to invade aquatic and wet environments, where the amount of oxygen and light can be variable, leads to a high selective pressure on genes involved in respiration and photosynthesis.

Molecular markers are often used to establish population genetic relationships through phylogenetic studies. Five chloroplasts (rps16, rpl16, trnL-trnF, trnL-CD, trnG) and two nuclear markers (ITS, waxy) were used in previous phylogeny studies of Ludwigia sp. [START_REF] Liu | Multi-locus phylogeny of Ludwigia (Onagraceae): insights on infra-generic relationships and the current classification of the genus[END_REF].

However, no SSR markers had previously been made available for the Ludwigia genus, or more broadly, the Onagraceae. In this study, we identified 45 to 65 SSR markers depending on the Ludwigia species. Most of them were AT mononucleotides, as already recorded for other angiosperms [START_REF] Maheswari | Chloroplast genome analysis of Angiosperms and phylogenetic relationships among Lamiaceae members with particular reference to teak (Tectona grandis L.f)[END_REF][START_REF] Zhang | The Complete Chloroplast Genome Sequences of Five Epimedium Species: Lights into Phylogenetic and Taxonomic Analyses[END_REF]. In addition, we identified various genes with highly mutated regions that can also be used as SNP markers. Chloroplast SSRs (cpSSRs) represent potentially useful markers showing high levels of intraspecific variability due to the non-recombinant and uniparental inheritance of the plastomes [START_REF] Huang | Development of high transferability cpSSR markers for individual identification and genetic investigation in Cupressaceae species[END_REF]100]. Chloroplast SSR characteristics for Ludwigia sp. (location, type of SSR) were similar to those described in most plants. While the usual molecular markers used for phylogenetic analysis are nuclear DNA markers, cpSSRs have also been used to explore cytoplasmic diversity in many studies [101-103]. To conclude, the 13 highly variable loci and cpSSRs identified in this study are potential markers for population genetics or phylogenetic studies of Ludwigia species, and more generally, Onagraceae.

Conclusions

In this study, we conducted the first-time sequencing and assembly of the complete plastomes of Lpm and Lgh, which are the only available genomic resources for functional analysis in both species. We were able to identify the existence of two haplotypes in both Lpm and Lgh, while the absence of the Lo genome precluded further investigation for this species. Comparison of all 10 Onagraceae plastomes revealed a high degree of conservation in genome size, gene number, structure, and IR boundaries. However, to further elucidate the phylogenetic analysis and evolution in Ludwigia and Onagraceae, additional chloroplast genomes will be necessary, as highlighted in recent studies of Iris and Aristidoideae species [104].

Figures

(

  Michx.) Greuter & Burdet (1987) subsp. hexapetala (Hook. & Arn.) (Nesom & Kartesz 2000)

[

  WLTDIAHHHLAIA] which corresponds to a region predicted as transmembrane. We then observe three direct repeats conserved in Lpm and Lgh in ycf1, accD and clpP1 respectively, two unique DRs in Lo (in the accD gene and rps12-clpP1 intergene) and one in Lgh (in the clpP1 intron 1 and clpP1 intron 2).

copies of 9

 9 amino acids [DESENSNEE] in Lgh and Lpm, two of which form a larger duplication of 17 aa [FLSDSDIDDESENSNEE]. Similarly, the TRs present only in Lo generate two perfect 9 amino acid repeats [EELSEDGEE], included in two longer degenerate repeats of 27 nt (Add.

Figure 1 :

 1 Figure 1: Two structural haplotypes of L. grandiflora plastomes representing the flip-flop organization of SSC segment.

Figure 2 :

 2 Figure 2: Comparative results of L. grandiflora short read (SR) assemblies. A: Total number of contigs obtained with the uncorrected (dark green) and corrected (light green) chloroplast SRs for the 4 assemblers (ABySS, MEGAHIT, Velvet and SPAdes). B: Comparison of the size of contigs assembled by the 4 tools using corrected or uncorrected SRs. C: Boxplot showing the distribution of these contigs by size and the improvement brought by the prior correction of the SRs with the long reads for each tool.

Figure 3 :

 3 Figure 3: Circular representation of annotations plastomes in Ludwigia octovalis, Ludwigia grandiflora and Ludwigia peploides using ogdraw. Each card contains four circles. From the center outwards, the first circle shows forward and reverse repeats (red and green arcs, respectively). The next circle shows tandem repeats as bars. The third circle shows the microsatellite sequences. Finally, the fourth and fifth circles show the genes colored according to their functional categories (see colored legend). Only the haplotype 1 of L. grandiflora is represented as haplotype 2 only diverge by the orientation of the SSC segment.

Figure 4 :

 4 Figure 4: Comparison of the borders of LSC, SSC, and IR regions in Onagraceae plastomes. A: Comparison of the junction between large single-copy (LSC, light blue), inverted repeat (IR, orange) and short single-copy (SSC, light green) regions among the chloroplast genomes of L. octovalvis, L. peploides and L. grandiflora (both haplotypes). Genes are denoted by colored boxes and the gaps between genes and boundaries are indicated by base lengths (bp). JLB: junction line between LSC and IRb; JSB: junction line between IRb and SSC; JSA: junction line between SSC and IRa; JLA: junction line between IRa and LSC. B: Comparison of SSC boundaries in haplotype 1 (L. peploides and L. grandiflora haplotype 1) and haplotype 2 (L. octovalvis and L. grandiflora haplotype 2) plastomes.

Figure 5 :

 5 Figure 5: Comparison of LSC, SSC and IR regions boundaries in Onagraceae chloroplast genomes.Representative sequences from each genus have been chosen (noted R on the diagram) except for Oenothera lindheimeri (only 89.35 % identity with others Oenothera), Circaea alpina (99.5 % identity but all others Circaea are 99.9% identical) and Chamaenerion conspersum (99% but all others Chamaenerion are ca. 99.7 identical). As shown in Figure7, the 3 Ludwigia plastomas had the same structure, L. octovalvis was chosen as a representative of this genus.

Figure 6 :

 6 Figure 6: Illustration of nucleotide diversity of the three Ludwigia chloroplast genome sequences. The graph was generated using DnaSP software version 6.0 (windows length: 800 bp, step size: 200 bp). The x-axis corresponds to the base sequence of the alignment, and the y-axis represents the nucleotide diversity (π value). LSC, SSC and IR segments were indicated under the line representing the genes coding the proteins (in light blue) the tRNAs (in pink) and the rRNAs (in red). The genes marking diversity hotspots are noted at the top of the peaks.

Figure 7 :

 7 Figure 7:The Ka/Ks ratios of the 80 protein-coding genes of Ludwigia sp. plastomes. The blue curve represents L. grandiflora versus L. peploides, purple curve denotes L. grandiflora versus L. octovalvis and green curve L. peploides versus L. octovalvis. Four genes (matK, accD, ycf2 and ccsA) have Ka/Ks ratios greater than 1.0, whereas the Ka/Ks ratios of the other genes were less than 1.0.

Figure 8 : 2 :Figure 3 :Add. Figure 4 :Figure 5 :

 82345 Figure 8: Phylogenetic tree based on the Ludwigia matK marker. Only four sequences are complete (499 aa, yellow star), the others correspond to barcode amplification ranging from 128 to 386 aa, with an average of 244 aa.

Figure 6 : 7 :BAdd. Figure 8 :

 678 Diagram showing the position of tandem repeats in the accD gene. L. octovalis (in red) and L. peploides and L. grandiflora (in green). We also observe the consequences of these repetitions on the insertion of amino acids, also repeated. Comparison of the three Ludwigia plastomes using mVISTA, with the L. octovalvis as a reference. A: The y-axis represents the identity percentage (between 50 and 100%). The arrows show the genes (in green: proteins genes, in purple: rRNAs and in fuchsia: tRNAs). Blue blocks indicate exonic regions. LCS, IR and SSC are also distinguished (in dark blue, red and green, respectively). The second line corresponds to L. grandiflora haplotype 2 (For this haplotype, SSC segment is oriented like L. octovalvis) and the third line corresponds to L. peploides for which the SSC region has been artificially oriented in the same way as the two other plastomes to allow comparison. B: Small box showing a part of the alignment and presenting the consequences if we do not artificially orient the SSC segments in the same direction for the analysis.A Lollipop diagram allowing the visualization of SNPs and their translational effects on the ycf2. A: localization of the 256 single nucleotide polymorphisms (SNP) observed by comparing L. grandiflora-L. peploides with L. octovalvis. Two regions particularly dense in SNPs (between 3420 and 3460 and between 6100 and 6600) have been zoomed into to allow better reading. B: Effect of these SNPs on the translated sequence of L. octovalvis, compared to Ycf2 of the other two species: non conservative mutation: red square; conservative mutation: circle green; deletion: triangle_point_up blue and insertion: triangle_point_down, orange. As for A, two regions were zoomed into in order to distinguish each mutation.

  Photosynthesis

  -UAC trnE-UUC-trnT-GGU Common palindromic and quasi-palidromic repeats Locus Comment in L. octovalvis in L. grandiflora and L. peploides TTTAACGAATATTAATATT t GTTAAA TTAA c GAATATTAATATTCTTTAA TTTAACGAATATTAATATTCGTTAAA TTAAAGAATATTAATATTCTTTAA trnR-UCU-atpA trnR-UCU-atpA Full deletion in L. octovalvis , 6 bases deletion in L. grandiflora L. octovalvis ATTGAAATTCGAATTTCAAT psbZ-trnG-GCC Full deletion in L. grandiflora and L. peploides L. peploides and L.

Table 1 .

 1 The general characteristics of the three Ludwigia plastomes

		A	sample.gr					3430	3431	3432	3433	3434	3435	3436	3437	3438	3439	3440	3441	3442	3443	3444	3445	3446	3447	3499
			3400				3420										3440		TD1	TD3	3460	3480	3500
	sample.gr	368	385 458	1252	1339	1340 1342 1343 1615 1630			2701 2702 2703 2981 3106 3430 3431 3432 3433 3434 3435 3436 3437 3438 3439 3440 3441 3442 3443 3444 3445 3446 3447 3499 3521 3714 3917 4060 4061 4105 4147 4170 4461 4469 4559 4627 4706 4707 4708 4739 4741 4961 4964 4965 4966 4967 5186 5195 5206 5300 5446 5582 5593 5626 5629 5654 5744 6051 6064 6086 6087 6088 6089 6090 6091 6092 6093 6094 6095 6096 6097 6098 6099 6100 6227 6228 6229 6232 6233 6235 6239 6257 6261 6274 6275 6276 6277 6278 6279 6280 6281 6282 6284 6286 6288 6291 6292 6298 6300 6308 6320 6326 6334 6338 6361 6414 6427 6488 6493 6542 6552 6556 6572 6578 6601 6602 6621 6741
			1000			2000	TD1		TD2 3000	TD3		TD4 4000	TD5	5000	6000
	sample.gr	6051 6064 6086 6087 6088 6089 6090 6091 6092 6093 6094 6095 6096 6097 6098 6099 6100	6227 6228 6229 6232 6233 6235 6239 6257 6261 6274 6275 6276 6277 6278 6279 6280 6281 6282 6284 6286 6288 6291 6292 6298 6300 6308 6320 6326 6334 6338 6361 6414 6427	6488	6493	6542 6552	6556 6572	6578
			6100				6200							6300	TD1			6400	6500	6600
		B

Table 2 :

 2 Genes present in the plastome of Ludwigia sp.

	L.octovalvis*	L. grandiflora subsp.	L. peploides subsp.
					hexapetala	montevidensis
			Size (bp)
	159;396			159;584	159;537
	LSC	90;183			90;272	90;156
	SSC	19;703			19;788	19;799
	IR	24;755			24;762	24;791
			GC%
		37;4			37;3	37;3
	LSC	35;2			35;1	35;1
	SSC	32			31;7	31;7
	IR	43;5			43;5	43;4
	Function			Name
	Rubisco		rbcL
	Photosystem I (PSI)		psaA; psaB; psaC; psaI; psaJ
	PSI assembly factors	ycf3 # (pafI); ycf4 (pafII)
	Photosystem II		psbA; psbB; psbC; psbD; psbE; psbF; psbH; psbI; psbJ; psbK; psbL; psbM; pbf1 (psbN) psbT; psbZ
	ATP synthase		atpA; atpB; atpE; atpF # ; atpH; atpI
	Cytochrome b6f petA; petB Cytochrome biogenesis ccsA
	NADPH dehydrogenase	ndhA	# ; ndhB**
	Transcription		rpoA; rpoB; rpoC1 # ; rpoC2
	Small ribosomal proteins	rps2; rps3; rps4; rps7**; rps8; rps11; rps12**	# ; rps14; rps15; rps16 # ; rps18; rps19
	Large ribosomal proteins	rpl2**	# ; rpl14; rpl16 # ; rpl20; rpl22; rpl23**; rpl32; rpl33; rpl36
					1

* KX827312 (ref) # ; petD # ; petG; petL; petN # ; ndhC; ndhD; ndhE; ndhF; ndhG; ndhH; ndhI; ndhJ

Table 2 :

 2 Genes present in the plastome of Ludwigia sp.

Table 3B :

 3B Direct repeats detected on Ludwigia sp. plastomes Lo = Ludwigia octovalvis; Lgh = L. grandiflora subsp. Hexapetala; Lpm = L. peploides subsp. Montevidensis.

	Sequence	Lo	Lgh	Lpm Size (nt)	Spacers (nt)	Region	Locus	Comments
	TTCAATTGGAACGGACGATTCGTCAATCATCT				32	37	SSC	ycf1	2 copies. In Lo, one mutation (G->A) in the second copie
	CATCGATGATGAAAGTGAAAACAGTAATGAAGAGG	X			35	28 -22 -11	LSC	accD	3 perfects copies and 1 mutated (G->A) copie in Lgh and Lpm . Region of 174
	AGATGGTGAAGAACCTTATGAAGATGGTGAAGAACCTTATG		X	X	41	22	LSC	accD	nt deleted in Lo Region of 147 nt deleted in Lgh and Lpm
	TATCAAATCAACAATGCCATACCGTAGGGCAT		X	X	32	22 -21	LSC	rps12-clpP1	3 copies
	TTAAGAGCCGTACAGGCACCTTTTGATGCATACGG	X				408 in L.p , 406 in L. g	LSC	clpP1	2 copies. In Lgh, one mutation (C->T) in the second copie
	TTAAGAGCCGTACAGGCACTTTTTGATGCATACGG	X		X	35	811	LSC	clpP1 intron 1-intron 2
	TGCAATAGCCAAATGATGATGAGCAATATCAGTCAGCCATA				41	2178	LSC	psaB-psaA
	47

Table 3C :

 3C Palindromic repeats detected on Ludwigia sp. plastomes

	Common perfect palidromic repeats	Locus	Comment

Acknowledgements

We are grateful to Luis Portillo-Lemus for developing the high molecular weight genomic DNA extraction protocol. All sequencing experiments were performed at the PGTB (doi:10.15454/1.5572396583599417E12).

Declarations

An official authorization from "prefecture de Loire Atlantique" was obtained to collect and transport L. grandiflora subsp. hexapetala (Lgh) from the Mazerolles swamps (commune Petit Mars, France), and L. peploides subsp. montevidensis (Lpm) from La Musse (commune Saint Etienne de Montluc, France) (prefectorial decree n°2018/SEE/2423). In accordance with French legislation on invasive species and as indicated in prefectorial decree n°2018/SEE/2423 (article 3), the samples were duly destroyed and under no circumstances could they be placed in a herbarium, which is strictly forbidden regarding local and national guidelines.

The datasets generated and/or analysed during the current study were available in GenBank (for Lgh haplotype 1, (LGH1) OR166254 and Lgh haplotype 2, (LGH2) OR166255; for Lpm haplotype, (LPM) OR166256).

The authors declare that they have no competing interests.

The post-doctoral research grant of Anne-Laure Le Gac was supported by the Conseil regional Bretagne (SAD18001).

Additional Figures

Add. Figure