Delphine Giraud
email: delphine.giraud@univ-rennes1.fr

Oscar Lima
email: oscar.lima@univ-rennes1.fr

Virginie Huteau
email: virginie.huteau@inrae.fr

Olivier Coriton
email: olivier.coriton@inrae.fr

Julien Boutte
email: boutte.julien@gmail.com

Ales Kovarik
email: kovarik@ibp.cz

Andrew R Leitch
email: a.r.leitch@qmul.ac.uk

Ilia J Leitch
email: i.leitch@kew.org

Malika Aïnouche
email: malika.ainouche@univ-rennes1.fr

Armel Salmon
email: armel.salmon@univ-rennes1.fr

Evolutionary dynamics of transposable elements and satellite DNAs in polyploid Spartina species

Keywords: genome dynamics, polyploidy, transposable elements, satellite DNA, Spartina

 First repeat annotations from high-throughput genome sequencing in polyploid Spartina  Repeat dynamics among Spartina species is correlated with phylogenetic relationships  Genome evolution is determined by divergence times rather than by ploidy levels.  Repeat patterns of the tetraploid S. spartinae suggest relationships with hexaploids

Introduction

Repetitive sequences, including dispersed repeats such as transposable elements (TEs), or tandem repeats such as satellite DNA, represent an important fraction of plant genomes that impact evolutionary dynamics [START_REF] Oliver | Transposable elements: powerful facilitators of evolution[END_REF][START_REF] Bennetzen | The contributions of transposable elements to the structure, function, and evolution of plant genomes[END_REF][START_REF] Mehrotra | Repetitive sequences in plant nuclear DNA: types, distribution, evolution and function[END_REF]. Together with polyploidy (whole genome duplication), repetitive sequences are the key drivers of genome size variation in plants, where up to c. 2400-fold variation may be observed [START_REF] Pellicer | The largest eukaryotic genome of them all?[END_REF][START_REF] Pellicer | Genome size diversity and its impact on the evolution of land plants[END_REF][START_REF] Dodsworth | Genome size diversity in angiosperms and its influence on gene space[END_REF][START_REF] Michael | Plant genome size variation: bloating and purging DNA[END_REF][START_REF] Ren | A mechanism for genome size reduction following genomic rearrangements[END_REF][START_REF] Soltis | Polyploidy and genome evolution in plants[END_REF]. In angiosperms, the repeat content, much of which comprises TEs, ranges from 18% in Arabidopsis thaliana to 80% in Triticum aestivum and 85% in Zea mays [10][START_REF] Tenaillon | A triptych of the evolution of plant transposable elements[END_REF][START_REF] Oliver | Transposable elements: powerful contributors to Angiosperm evolution and diversity[END_REF][START_REF] Kim | The genomes and transposable elements in plants: are they friends or foes?[END_REF][START_REF] Wicker | Impact of transposable elements on genome structure and evolution in bread wheat[END_REF]. These elements are classified according to their transposition mode: retrotransposons (or Class I elements) replicate via a "copy-paste" process (resulting in genome size variation), while DNA transposons (or Class II elements) replicate via a "cut-paste" process [START_REF] Kumar | Plant retrotransposons[END_REF][START_REF] Feschotte | DNA transposons and the evolution of eukaryotic genomes[END_REF][START_REF] Wicker | A unified classification system for eukaryotic transposable elements[END_REF]. Transposable elements may accumulate in various coding and non-coding genomic regions following transposition bursts, and may be subject to differential expansion or loss during species evolution [START_REF] Oliver | Transposable elements: powerful facilitators of evolution[END_REF][START_REF] Vitte | LTR retrotransposons in rice (Oryza sativa, L.): recent burst amplifications followed by rapid DNA loss[END_REF][START_REF] Civáň | On the coevolution of transposable elements and plant genomes[END_REF][START_REF] Staton | The sunflower (Helianthus annuus L.) genome reflects a recent history of biased accumulation of transposable elements[END_REF][START_REF] Baidouri | Comparative genomic paleontology across plant kingdom reveals the dynamics of TE-driven genome evolution[END_REF][START_REF] Wang | Transposable elements play an important role during cotton genome evolution and fiber cell development[END_REF]. Thus, they play an important role in the dynamics of expansion and contraction of plant genomes that may be observed even between closely related species [START_REF] Leitch | Genomic plasticity and the diversity of polyploid plants[END_REF][START_REF] Renny-Byfield | Doubling down on genomes: polyploidy and crop plants[END_REF][START_REF] Wendel | Evolution of plant genome architecture[END_REF][START_REF] Vicient | Impact of transposable elements on polyploid plant genomes[END_REF]. Transposable elements not only shape genome structure, but can also substantially affect gene expression, when inserted in coding or regulatory regions [START_REF] Kashkush | Transcriptional activation of retrotransposons alters the expression of adjacent genes in wheat[END_REF][START_REF] Lisch | How important are transposons for plant evolution?[END_REF][START_REF] Pereira | The role of transposable elements in the evolution of aluminium resistance in plants[END_REF]. To prevent TE proliferation and their putative consequences on gene regulation and function, TE expression is controlled by several epigenetic silencing pathways, such as mRNA cleavage, small RNA synthesis and DNA methylation (via transcriptional and post-transcriptional gene silencing) [START_REF] Vicient | Transcriptional activity of transposable elements in maize[END_REF][START_REF] Axtell | Classification and comparison of small RNAs from plants[END_REF][START_REF] Fultz | Silencing of active transposable elements in plants[END_REF][START_REF] Sigman | The first rule of plant transposable element silencing: location, location, location[END_REF].

A notable feature that has emerged from comparative studies of TE activity is that TE amplification does not appear to be constant, with some TE lineages showing evidence of periods of transposition activity, alternating with periods of relative quiescence [START_REF] Vicient | Impact of transposable elements on polyploid plant genomes[END_REF][START_REF] Macas | In depth characterization of repetitive DNA in 23 plant genomes reveals sources of genome size variation in the legume tribe Fabeae[END_REF]. Several studies have suggested that TE activity is influenced by environmental factors, such as biotic or abiotic stress [START_REF] Kalendar | Genome evolution of wild barley (Hordeum spontaneum) by BARE-1 retrotransposon dynamics in response to sharp microclimatic divergence[END_REF][START_REF] Cavrak | How a retrotransposon exploits the plant's heat stress response for its activation[END_REF][START_REF] Grandbastien | LTR retrotransposons, handy hitchhikers of plant regulation and stress response[END_REF][START_REF] Ito | A stress-activated transposon in Arabidopsis induces transgenerational abscisic acid insensitivity[END_REF].

Allopolyploidy, which combines interspecific hybridization with polyploidy, is widespread in plants. Both the merging of the different species genomes into one nucleus and polyploidy event itself have the potential to J o u r n a l P r e -p r o o f impact TE regulatory processes [START_REF] Vicient | Impact of transposable elements on polyploid plant genomes[END_REF][START_REF] Mcclintock | The significance of responses of the genome to challenge[END_REF][START_REF] Parisod | Impact of transposable elements on the organization and function of allopolyploid genomes[END_REF], although it remains unclear which process has the most significant effect on TE activity. Altered patterns of epigenetic and TE transcriptional activities from expectation (i.e. the sum of the activity seen in the diploid parents) have been observed in both experimentally resynthesized hybrids and allopolyploids (e.g. Arabidopsis [START_REF] Madlung | Genomic changes in synthetic Arabidopsis polyploids[END_REF][START_REF] Göbel | Robustness of transposable element regulation but no genomic shock observed in interspecific Arabidopsis hybrids[END_REF], wheat [START_REF] Kashkush | Transcriptional activation of retrotransposons alters the expression of adjacent genes in wheat[END_REF][START_REF] Yaakov | Mobilization of Stowaway-like MITEs in newly formed allohexaploid wheat species[END_REF], tobacco [START_REF] Petit | Differential impact of retrotransposon populations on the genome of allotetraploid tobacco (Nicotiana tabacum)[END_REF][START_REF] Petit | Mobilization of retrotransposons in synthetic allotetraploid tobacco[END_REF]) and natural hybrids and allopolyploids [START_REF] Beaulieu | The allotetraploid Arabidopsis thaliana-Arabidopsis lyrata subsp. petraea as an alternative model system for the study of polyploidy in plants[END_REF][START_REF] Parisod | Rapid structural and epigenetic reorganization near transposable elements in hybrid and allopolyploid genomes in Spartina[END_REF]. Most of these studies suggest that the immediate effects of allopolyploidy have the most impact on TE regulatory processes, potentially leading to bursts of TE activity, although disrupted patterns may stabilize within a few generations [START_REF] Ha | Small RNAs serve as a genetic buffer against genomic shock in Arabidopsis interspecific hybrids and allopolyploids[END_REF]. Nevertheless, allopolyploids of different ages (i.e. recent to millions of years), show evidence for differential TE amplification and elimination [START_REF] Hu | The history and disposition of transposable elements in polyploid Gossypium[END_REF][START_REF] Sarilar | a Stowaway MITE from Brassica: recently active copies preferentially accumulate in the gene space[END_REF][START_REF] Renny-Byfield | Diploidization and genome size change in allopolyploids is associated with differential dynamics of low-and high-copy sequences[END_REF][START_REF] Senerchia | Contrasting evolutionary trajectories of multiple retrotransposons following independent allopolyploidy in wild wheats[END_REF][START_REF] Cheng | Comparative effect of allopolyploidy on transposable element composition and gene expression between Gossypium hirsutum and its two diploid progenitors[END_REF]. It might be expected that genome redundancy in polyploids would buffer against the mutagenic impact of TE activity [START_REF] Civáň | On the coevolution of transposable elements and plant genomes[END_REF][START_REF] Vicient | Impact of transposable elements on polyploid plant genomes[END_REF][START_REF] Parisod | Impact of transposable elements on the organization and function of allopolyploid genomes[END_REF]. The short-term responses of allopolyploid genomes to different TE composition inherited from their parental species is also expected to influence subgenome dominance and the fractionation process which contribute to the longterm divergence of polyploids [START_REF] Edger | Subgenome dominance in an interspecific hybrid, synthetic allopolyploid, and a 140-year-old naturally established neo-allopolyploid monkeyflower[END_REF][START_REF] Bottani | Gene expression dominance in allopolyploids: hypotheses and models[END_REF].

The genus Spartina Schreb (Poaceae) offers excellent opportunities for comparative genomic analyses, enabling patterns and tempos of repetitive sequence evolution to be deciphered. Since the last whole genome duplication event (WGD) in the ancestry of family Poaceae [START_REF] Van De Peer | The evolutionary significance of polyploidy[END_REF][START_REF] Zwaenepoel | Inference of ancient whole-genome duplications and the evolution of gene duplication and loss rates[END_REF](Van de Peer et al 2017, Zwaenepoel and Van de Peer 2019) polyploidy events in Spartina have given rise to 15 species, which range from tetraploid to dodecaploid, with evidence of extensive reticulate evolution in the species' ancestries (resulting from hybridization, introgression and allopolyploidy) [START_REF] Marchant | Evolution in Spartina (Gramineae): III. Species chromosome numbers and their taxonomic significance[END_REF][START_REF] Baumel | Genetic evidence for hybridization J o u r n a l P r e -p r o o f between the native Spartina maritima and the introduced Spartina alterniflora (Poaceae) in South-West France: Spartina x neyrautii re-examined[END_REF][START_REF] Ayres | Hybridization between invasive Spartina densiflora (Poaceae) and native S. foliosa in San Francisco Bay, California, USA[END_REF][START_REF] Ainouche | Hybridization, polyploidy and invasion: lessons from Spartina (Poaceae)[END_REF][START_REF] Renny-Byfield | Flow cytometry and GISH reveal mixed ploidy populations and Spartina nonaploids with genomes of S. alterniflora and S. maritima origin[END_REF]. No diploid Spartina species are known. Species in Spartina are perennial and mostly colonize coastal salt marshes, where they are considered as "ecosystem engineers" because they increase sediment accretion. Spartina species are particularly tolerant to salt stress, drought, anoxic conditions and pollution by heavy metals and hydrocarbons [START_REF] Bedre | Transcriptome analysis of smooth cordgrass (Spartina alterniflora Loisel), a monocot halophyte, reveals candidate genes involved in its adaptation to salinity[END_REF][START_REF] Paredes-Páliz | Modulation of Spartina densiflora plant growth and metal accumulation upon selective inoculation treatments: a comparison of gram negative and gram positive rhizobacteria[END_REF][START_REF] Robertson | Genetic and epigenetic variation in Spartina alterniflora following the Deepwater Horizon oil spill[END_REF][START_REF] Alvarez | Transcriptome response of the foundation plant Spartina alterniflora to the Deepwater Horizon oil spill[END_REF][START_REF] Cavé-Radet | Increased tolerance to organic xenobiotics following recent allopolyploidy in Spartina (Poaceae)[END_REF].

Phylogenetic analyses have revealed that Spartina forms a distinctive clade within genus Sporobolus, rendering Sporobolus polyphyletic [START_REF] Peterson | A molecular phylogeny and new subgeneric classification of Sporobolus (Poaceae: Chloridoideae: Sporobolinae)[END_REF]. Patterns of branching within the phylogenetic trees indicate that Spartina diversified within the last 12-20 million years [START_REF] Peterson | A molecular phylogeny and new subgeneric classification of Sporobolus (Poaceae: Chloridoideae: Sporobolinae)[END_REF][START_REF] Baumel | Molecular phylogeny of hybridizing species from the genus Spartina Schreb. (Poaceae)[END_REF][START_REF] Ainouche | Hybridization, polyploidy and speciation in Spartina (Poaceae)[END_REF][START_REF] Fortune | Evolutionary dynamics of Waxy and the origin of hexaploid Spartina species (Poaceae)[END_REF][START_REF] Rousseau-Gueutin | The chloroplast genome of the hexaploid Spartina maritima (Poaceae, Chloridoideae): comparative analyses and molecular dating[END_REF] and comprises two main subclades, that diverged 6-10 million years ago [START_REF] Rousseau-Gueutin | The chloroplast genome of the hexaploid Spartina maritima (Poaceae, Chloridoideae): comparative analyses and molecular dating[END_REF]: (i) a tetraploid lineage (2n=4x=40) including New World species, and (ii) a hexaploid lineage (2n=6x=60, 62) which includes the North-American S. alterniflora, S. foliosa and the European species S. maritima (Fig. 1 and Table 1). The phylogenetic affinities of the South American tetraploid species S. spartinae (syn. S. argentinensis) remains unresolved, being placed alternatively as sister to the hexaploid lineage or sister to all other Spartina species [START_REF] Baumel | Molecular phylogeny of hybridizing species from the genus Spartina Schreb. (Poaceae)[END_REF][START_REF] Rousseau-Gueutin | The chloroplast genome of the hexaploid Spartina maritima (Poaceae, Chloridoideae): comparative analyses and molecular dating[END_REF]. Hybridization within and between these subclades has given rise to additional ploidy levels (7x, 8x, 9x, 12x, including a classical example of neopolyploid speciation reviewed in [START_REF] Ainouche | Polyploid evolution in Spartina: dealing with highly redundant hybrid genomes[END_REF].

Cytogenetic investigations reveal that Spartina genome sizes (estimated using flow cytometry) vary between and within ploidy levels. For example, amongst tetraploids (2n=40), genome sizes vary from 1.45 pg/2C in S. bakeri [START_REF] Fortune | The enigmatic invasive Spartina densiflora : a history of hybridizations in a polyploidy context[END_REF] to 1.56 pg/2C in S. pectinata [START_REF] Kim | Cytogeographic distribution and genome size variation in prairie cordgrass (Spartina pectinata Bosc ex Link)[END_REF] and 2.00 pg/2C in S. spartinae [START_REF] Baisakh | Development and interspecific transferability of genic microsatellite markers in Spartina spp. with different genome size[END_REF]. These data reveal lineage-specific genome size changes have occurred in the recent evolutionary history of these polyploid species.

To date, no exhaustive investigations have been undertaken to evaluate the nature and dynamics of repetitive sequences between different species of Spartina that diversified since tetra-and hexa-mesoploid events in the last 6-10MYs. Instead, previous studies have examined the dynamics of targeted TEs following recent allopolyploidy. For example, using Transposon-display approaches [START_REF] Baumel | Retrotransposons and genomic stability in populations of the young allopolyploid species Spartina anglica C.E. Hubbard (Poaceae)[END_REF] it was shown that there have J o u r n a l P r e -p r o o f been no transposition burst associated with the divergence of the neoallododecaploid S. anglica from its hexaploid parental species S. alterniflora and S. maritima. In contrast, methylation-sensitive AFLP analyses revealed that hybridization (in the F1 sterile hybrid S. x townsendii), was accompanied by substantial methylation changes, mostly in regions flanking TEs [START_REF] Parisod | Rapid structural and epigenetic reorganization near transposable elements in hybrid and allopolyploid genomes in Spartina[END_REF], that were inherited in the formation of S. anglica [START_REF] Salmon | Genetic and epigenetic consequences of recent hybridization and polyploidy in Spartina (Poaceae)[END_REF].

In this paper, we take advantage of high-throughput (Illumina) genome sequencing to explore the evolutionary dynamics of repetitive sequences in tetraploid and hexaploid Spartina species with contrasting genome sizes. Of particular interest to this analysis is that some of these species have been involved in the formation of recent hybrids and neoallopolyploids. We first evaluate the relative amounts of the repetitive compartment in the different polyploid lineages, and determine what sequence types account for genome size variation among species. This allowed us to examine whether the differential evolution of repetitive sequences is correlated with ploidy levels, phylogenetic relationships, or the known divergence times between lineages.

Material and methods

Plant material

Five Spartina species were selected for analysis representing the tetraploid (4x) and hexaploid (6x) parental lineages that gave rise to higher ploidy levels (7x, 8x, 9x 12x) observed in the genus [START_REF] Ainouche | Polyploid evolution in Spartina: dealing with highly redundant hybrid genomes[END_REF]. The two sister hexaploid species S. maritima (Curtis) Fernald (2n=6x=60; Fig. 1, see Table 1 for synonyms and nomenclature) and S. alterniflora Loisel. (2n=6x=62) were selected for analysis because of their involvement in the recent (i.e. < c. 130 years ago) allopolyploid speciation event that gave rise to S. anglica [START_REF] Marchant | Evolution in Spartina (Gramineae): III. Species chromosome numbers and their taxonomic significance[END_REF][START_REF] Baumel | Molecular phylogeny of hybridizing species from the genus Spartina Schreb. (Poaceae)[END_REF][START_REF] Fortune | Evolutionary dynamics of Waxy and the origin of hexaploid Spartina species (Poaceae)[END_REF][START_REF] Baumel | Molecular investigations in populations of Spartina anglica C.E. Hubbard (Poaceae) invading coastal Brittany (France)[END_REF]. Three tetraploid species (2n=4x=40) were also selected. These being two closely related-species S. versicolor (Fabre) and S. bakeri Merr., that diverged recently from each other (less than 2 million years ago (Mya), Fig. 1). A third, more distantly related tetraploid species, S. spartinae (Trin.) Merr., was studied because it exhibits the highest tetraploid genome size reported so far (1.97pg/2C, based on our own genome size estimate, see below).

The hexaploid species were sampled along the French Atlantic coast from their natural environment: S. maritima on the Etel river marshes (Presqu'île du Verdon, Morbihan, France) and S. alterniflora on the Faou river marshes (Le Faou, Finistère, France). S. maritima and S. alterniflora were also sampled in Southern England to detect putative variations in repeat accumulation between populations of the same species. S. maritima was collected at the Isle of Wight (UK) and S. alterniflora at Marchwood (UK). The tetraploid S.

versicolor was collected on the Mediterranean coast at Hyères (Var, France), S. bakeri in Florida (Miami Botanical Garden, USA) and S. spartinae in the Santa Fe Province (Argentina). The plants were maintained in the greenhouse at the University of Rennes 1 before analysis.

Flow cytometry

The ploidy level and genome size of each species was assessed using flow cytometry at the Imagerie-Gif facilities (Institute for Integrative Biology of the Cell, UMR 9198 CNRS-CEA, Gif sur Yvette, France) using propidium iodide (50µg/ml) to stain the nuclei following established protocols [START_REF] Fortune | The enigmatic invasive Spartina densiflora : a history of hybridizations in a polyploidy context[END_REF]. Briefly, fresh leaves were chopped in buffer containing 45mM MgCl2, 30mM sodium-citrate, 60mM MOPS (pH 7.0), 1% PVP-10, 0.1% Triton X-100 and 10mM sodium metabisulfite (S2O5Na2). Petunia hybrida (2.85pg/2C) was used as the internal J o u r n a l P r e -p r o o f calibration standard and the samples were analyzed on a CytoFLEX S flow cytometer (Beckman Coulter).

Genome size measurements were conducted on the same plants that were used for sequencing.

Sequencing and de novo identification of repeated sequences

Repeated sequences for each species were de novo retrieved from Illumina genomic sequencing data.

Genomic DNA was extracted from fresh leaves and Illumina libraries were prepared according to the Tru-Seq PCR-Free Protocol provided by Illumina. Libraries were sequenced (100-150 bp; paired-end with 500 bpinserts) at the BGI (Beijing Genomics Institute) using the Hi-Seq X technology with a read depth ranging from 20x to 90x depending on species. Random samples of 1 million Illumina paired-end reads (total of 2 million reads by sample) were processed using the RepeatExplorer pipeline [START_REF] Novák | Graph-based clustering and characterization of repetitive sequences in next-generation sequencing data[END_REF][START_REF] Novák | RepeatExplorer: a Galaxy-based web server for genome-wide characterization of eukaryotic repetitive elements from next-generation sequence reads[END_REF] to identify repeated sequences in each species in the absence of an assembled reference genome. This approach uses a similarity-based clustering of reads to build consensus sequences representing different types of repeated sequences. In S. alterniflora, the presence of satellite DNA sequences increased the computational and memory requirements for data analysis.

Consequently, a random sampling of reads was performed on the 2 million reads sample and only a subset of 854 068 reads was analyzed by RepeatExplorer for this species (Table 2).

Prior to RepeatExplorer analyses, all reads deriving from chloroplast DNA and rDNA were removed from the samples. Reads of each species were aligned or mapped on their respective rDNA or chloroplast genome (previously assembled by [START_REF] Boutte | Haplotype detection from next-generation sequencing in high-ploidylevel species: 45S rDNA gene copies in the hexaploid Spartina maritima[END_REF] [START_REF] Boutte | Haplotype detection from next-generation sequencing in high-ploidylevel species: 45S rDNA gene copies in the hexaploid Spartina maritima[END_REF] and Rousseau-Gueutin et al. 2015 [START_REF] Rousseau-Gueutin | The chloroplast genome of the hexaploid Spartina maritima (Poaceae, Chloridoideae): comparative analyses and molecular dating[END_REF]) using BLAST+ (v.2.5.0; parameters: blastn with 90% identity along 90% of the reads length; [START_REF] Altschul | Basic local alignment search tool[END_REF]) and Bowtie2 (parameters: --score-min, local, G, 52, 8; [START_REF] Langmead | Fast gapped-read alignment with Bowtie 2[END_REF]). Reads with more than 90% identity with chloroplast or ribosomal sequences were not used in the analyses described below.

Each sample of reads was analyzed independently with the RepeatExplorer pipeline using the Galaxy-web interface. All-to-all comparisons of reads and clustering were performed with default parameters (i.e. minimum of 40 nt overlap between reads with at least 80% identity in the overlapping region). Only clusters containing more than 200 reads (> 0.01% of the sampled reads) were analyzed further.

Reads assigned to each cluster were assembled with CAP3 [START_REF] Huang | CAP3: a DNA sequence assembly program[END_REF] (included in the RepeatExplorer pipeline) to obtain consensus sequences (contigs) representing different TE families. CAP3 contigs were then coassembled using Genome Assembler (Roche, v. 2.8; [START_REF] Margulies | Genome sequencing in microfabricated high-density picolitre reactors[END_REF]) with the same settings as CAP3 (i.e. min. overlap of 40 nt with at least 80% identity in the overlapping region) to improve the assembly process and maximize contig length. Finally, repeated sequence databases for each species were established consisting of contigs obtained after Genome Assembler co-assembling with non-co-assembled CAP3 contigs.

Repeated sequence annotation and quantification

Different methods were used to annotate consensus sequences of each database. First annotations were performed using RepeatMasker (v. 4.0.0; http://repeatmasker.org). During this step, reads of each cluster were aligned via rmblastn (v. 2.2.23+; [START_REF] Altschul | Basic local alignment search tool[END_REF] against "Viridiplantae" sequences of Repbase (database version at 08/2016; [START_REF] Jurka | Repbase Update, a database of eukaryotic repetitive elements[END_REF][START_REF] Bao | Repbase Update, a database of repetitive elements in eukaryotic genomes[END_REF]. To complete the annotation, assembled contigs were aligned with blastx against a TE-specific protein database (translation of Repbase "Viridiplantae" nucleic sequences into protein sequences). Satellite sequences were also identified with "self-to-self" sequence comparisons by carrying out dot-plot analyses using YASS [START_REF] Noe | YASS: enhancing the sensitivity of DNA similarity search[END_REF].

J o u r n a l P r e -p r o o f

For each species, the quantity of repeated sequences was estimated from the number of reads found within clusters representing different TE families (percentage of sampled reads). For validation of estimated abundances, a second evaluation was realized by mapping (i) the same sample of reads and (ii) a larger set of reads (approximately 330 millions reads per species) on the five Spartina repeated sequence databases using Bowtie2 (parameters: --score-min, local, G, 52, 8; [START_REF] Langmead | Fast gapped-read alignment with Bowtie 2[END_REF]). Comparisons of both quantifications are given in Supplementary Table S1.

Southern blot hybridization

The relative abundance of two main satellite sequences (see Results) was checked by Southern blot hybridization in S. alterniflora and S. maritima (both from English populations). Genomic DNAs were digested with restriction endonucleases (2x 5 U /ug DNA, for 3 h each), separated by agarose gel electrophoresis and transferred to Hybond XL membranes (GE-Healthcare, Little Chalfont, UK) using alkaline capillary transfer.

The probes were inserts of the Smar_CL1_1 and Salt_SALT28 clones. The Smar_CL_1 clone containing three units of a 167-bp satellite was obtained by PCR amplification of S. maritima DNA using primers derived from the S. maritima cluster 1 consensus sequence (Spartina_mar_sat_F: 5'CGGTCCCGTATATGAATTT3', Spartina_mar_sat_R: 5'GGAGTTTCGGTCAGTTTT3'). The Salt_SALT28 clone containing a single monomer of the 370-bp satellite was obtained by a classical method based on restriction digestion (BstNI) of S. alterniflora genomic DNA followed by gel separation, excision and purification of the 300-500 bp fraction. The fragments were ligated to a plasmid vector (pDrive, Qiagen) and clones screened by Southern hybridization with labeled S. alterniflora genomic DNA. Clones with strong signals were sequenced by the Sanger method using the SP6 primer. For labeling of probes, the inserts were amplified from plasmids using the universal Sp6 and T7 vector primers. About 100 ng of purified PCR product was radioactively labeled with 32 P-dCTP according to the manufacturer protocol (DecaLabel DNA Labeling Kit, Thermofisher, USA). Southern blot hybridization was carried out in a 0.25 M sodium phosphate buffer (pH 7.0) supplemented with 7% w⁄v sodium dodecyl sulphate (SDS) at 65℃. The membranes were washed with 2 x SSC (10 x SSC = 1.5 M NaCl, 0.15 M sodium citrate, pH 7.0), 0.1% SDS (twice for 5 min) and then with 0.2 x SSC and 0.1% SDS (twice for 15 min at 65℃). The membranes were exposed to a storage phosphor screen, scanned (Typhoon FLA7400, GEHealthcare), and the signal was quantified using Image Quant (GE-Healthcare).

Cytogenetic analyses

Cytogenetic analyses were conducted to determine the distribution of a highly repeated satellite sequence identified in the S. alterniflora genome (see Results). Primers were designed to amplify the repeat by PCR from S. alterniflora genomic DNA (5'-CCATGCGTGAGATGGTCATA and 5'-GCTTCTTCAGACTTGTGGGC).

The product was labeled using random priming with biotin-16-dUTP and used as a probe in Fluorescence in situ hybridization (FISH) experiments. In addition, the probe, pTa 71 [START_REF] Gerlach | Cloning and characterization of ribosomal RNA genes from wheat and barley[END_REF], which contains a 9-kb EcoRI fragment of the 35S rDNA repeat unit (18S-5.8S-26S genes and spacers) isolated from Triticum aestivum, was used as a control. This probe was labelled with Alexa-488 dUTP (Invitrogen, Life Technologies) by random priming.

Root tips from the five studied Spartina species were harvested and treated in the dark with 0.04% (w/v) 8-hydroxyquinoline for 2h at 4°C followed by 2 h at room temperature to accumulate metaphases. They were J o u r n a l P r e -p r o o f then fixed in 3:1 ethanol-glacial acetic acid for 12 h at 4°C and stored in ethanol 70 % at -20 °C. Before use, root tips were washed in 0.01 M citric acid-sodium citrate pH 4.5 buffer for 15 min and digested in a solution of 5% Onozuka R-10 cellulase 1µM (Sigma) and 1% Y23 pectolyase 1µM (Sigma) at 37 °C for 3 h 45 min.

The digested root tips were then carefully washed with distilled water for 30 min. One root tip was transferred to a slide and macerated with a drop of 3:1 fixative solution. The slides were dried at room temperature and stored at -20°C.

For FISH, chromosome preparations were incubated in RNase A (100ng/µL) and pepsin (0.05%) in 10 mM HCl, fixed with paraformaldehyde (4%), dehydrated in an ethanol series (70%, 90% and 100%) and airdried. The hybridization mixture consisted of 50% deionized formamide, 10% dextran sulfate, 2 X SSC, 1% SDS, 100 ng of labeled probe targeting the repeat and 50 ng of labelled pTa71 targeting 35S rDNA. The mixture was denatured at 92°C for 6 min and transferred to ice. Chromosomes were denatured in a solution of 70% (v/v) formamide in 2X SSC at 70°C for 2 min. The denatured probe was placed on the slide and in situ hybridization carried out overnight in a moist chamber at 37°C. After hybridization, slides were washed for 5 min in 50% formamide in 2X SSC at 42°C, followed by several washes in 4X SSC-Tween. The biotinylated probe was immunodetected using Texas Red avidin DCS (Vector Laboratories) and the signal was amplified with biotinylated anti-avidin D (Vector Laboratories). The chromosomes were mounted and counterstained in Vectashield (Vector Laboratories) containing 2.5 µg/mL 4',6-diamidino-2-phenylindole (DAPI). Fluorescence images were captured using a CoolSnap HQ camera (Photometrics, Tucson, Ariz) on an Axioplan 2 microscope (Zeiss, Oberkochen, Germany) and analyzed using MetaVueTM (Universal Imaging corporation, Downington, PA).

Phylogenetics analyses

The evolutionary history of the most abundant TEs identified in Spartina (i.e. Gypsy and Copia retrotransposons, LINE, CACTA -see below) were investigated using phylogenetic analyses of protein sequences coding for the reverse transcriptase (RT) domain of retrotransposons or the transposase domain of CACTA elements. Sequences were retrieved from each Spartina TE database using the DANTE tool in the Galaxy version of RepeatExplorer [START_REF] Novák | RepeatExplorer: a Galaxy-based web server for genome-wide characterization of eukaryotic repetitive elements from next-generation sequence reads[END_REF]. Consensus sequences which showed homology with RT domains were translated into proteins and retained when they represented at least 50% of the RT domain size (87 and 125 amino acids for Gypsy and Copia elements, respectively). Similar analyses were performed to select sequences in other species used as outgroups in the phylogenetic analysis. A set of RT sequences from Repbase (v. 23.08; [START_REF] Jurka | Repbase Update, a database of eukaryotic repetitive elements[END_REF][START_REF] Bao | Repbase Update, a database of repetitive elements in eukaryotic genomes[END_REF]) was selected for six species representing various plant lineages: Pinus taeda, Amborella trichopoda, Arabidopsis thaliana, Medicago truncatula, Zea mays and Oryza sativa. Only sequences without stop-codon were kept. Gypsy and Copia RT protein sequences were separately aligned using Clustal Omega [START_REF] Madeira | The EMBL-EBI search and sequence analysis tools APIs in 2019[END_REF].

Informative blocks in multiple sequence alignments were selected with the GBlocks package [START_REF] Castresana | Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis[END_REF]. Phylogenetic analyses were performed with the IQ-TREE software [START_REF] Nguyen | IQ-TREE: A fast and effective stochastic J o u r n a l P r e -p r o o f algorithm for estimating maximum-likelihood phylogenies[END_REF] which enabled the testing of different models of molecular evolution via ModelFinder [START_REF] Kalyaanamoorthy | ModelFinder: fast model selection for accurate phylogenetic estimates[END_REF] and the construction of maximum-likelihood trees. The best-fit substitution model used was LG+R6 (determined according to BIC criteria) and robustness of branches was estimated after 10 000 Ultrafast Bootstraps [START_REF] Hoang | Ufboot2: improving the ultrafast bootstrap approximation[END_REF].

Analyses of TE diversity

J o u r n a l P r e -p r o o f

Diversity within each TE lineage was estimated with two complementary methods. First, divergence between reads coding for RT (or transposase) was determined by pairwise sequence comparisons. All reads from a sample of each species were aligned on the consensus sequences encoding RT domains using Bowtie2 (parameters: --score-min, local, G, 52, 8; [START_REF] Langmead | Fast gapped-read alignment with Bowtie 2[END_REF]. The distance between each pair of aligned reads was then calculated under Kimura two parameters model [START_REF] Kimura | A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences[END_REF] with MEGA7 [START_REF] Kumar | MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets[END_REF]. Only distances between reads overlapping at least 50 nucleotides were considered.

Second, TE diversity was evaluated by detecting haplotypes using the pipeline previously developed [START_REF] Boutte | Reference transcriptomes and detection of duplicated copies in hexaploid and allododecaploid Spartina species (Poaceae)[END_REF]. This pipeline detects polymorphisms or SNPs (Single-Nucleotide Polymorphisms) from reads aligned to consensus TE sequences and constructs the different haplotypes based on shared and phased polymorphisms (setting parameters: min. read depth: 10; read-depth threshold for SNP detection: 10; min. number of shared SNP for haplotyping: 2). The number of haplotypes retrieved for each TE provides an estimation of the different TE copies.

Results

Annotation and quantitative evaluation of repeated sequences in Spartina genomes

Between 2.2-4.0% of reads (from samples of 2 million reads) showed sequence homology to plastid DNA, and 0.7-1.5% to ribosomal DNA, depending on the species (Table 2). Mapping of all Illumina read datasets to the Spartina repeated sequence databases confirmed that no sample bias was observed in the quantification of repeats by RepeatExplorer (see Supplementary Table S1).

RepeatExplorer forms clusters of reads that represent repeat sequences. The number of clusters (repeat types) that occupy > 1% of the genome ranged from 59 clusters in S. maritima to 74 clusters in S. bakeri (Table 2). In total, these clusters comprise repeated sequences that occupy more than 45% of the sampled genomes of both hexaploid species (Table 3). Amongst the tetraploids, while S. spartinae has a similar proportion of repeats (44% of the genome), the other two species have substantially lower proportions (33%). In all species, the most abundant TEs are Class I retrotransposons with the largest proportion of the repeat content being Gypsy (52-70% of total repeats) followed by Copia (16%-23%) elements (Table 3).

The two sister tetraploid species S. versicolor and S. bakeri have similar TE content predominantly comprising Gypsy elements belonging to Tekay and Ogre lineages (estimated to represent ~ 11% (74 Mb) and ~ 5.5% (37 Mb) of their genomes respectively), Copia elements in the SIRE lineage (~3.5% (24 Mb) of their genomes) and CACTA elements (~2.7% (18.5 Mb) of their genomes) (Fig. 2 and Supplementary Table S2).

Overall, the total repetitive fraction of the genomes (including satellite repeats) is very similar, comprising 224 Mb (33%) in S. versicolor and 230 Mb (33%) in S. bakeri. In contrast, S. spartinae, which is also tetraploid, contains double the amount of repetitive sequences (~ 421 Mb). This difference is explained by larger amounts of all TE families particularly the Ogre and Tekay lineages which are, respectively, three and two times more abundant in S. spartinae than in S. versicolor and S. bakeri. Nevertheless, given the larger genome size of S. spartinae (963 Mb/1C) compared with S. versicolor (680 Mb/1C) and S. bakeri (689 Mb/1C), the proportion of the S. spartinae genome occupied by repeats (i.e. 43.7%) is only a third higher.

As with the tetraploid species, the most abundant repeats in the hexaploids are LTR-retrotransposons (Fig. 2 and Supplementary Table S2). In total, Gypsy and Copia elements comprise 35-37% of their genomes, with differences only in the abundance of individual element types (Supplementary Table S2). For example, In addition to the variation in abundance of LTR-retrotransposons, other TE lineages also exhibit interspecific variation. For example, the non-LTR retrotransposon LINE-RTE is less abundant in the tetraploid species (~3 Mb) than in the hexaploid species (14 Mb in S. alterniflora and 21 Mb in S. maritima). Amongst the Class II transposons, CACTA elements represent a more substantial component of the S. maritima genome (50 Mb) compared with S. alterniflora (26 Mb) and the tetraploid genomes (~ 18-26 Mb) (Fig. 2 and Supplementary Table S2).

Three highly repeated satellites in Spartina

Three highly repeated satellite DNA sequences were identified in the Spartina genomes (Fig. 3). One of these repeats, which has a monomer unit length of 167 bp, occupies less than 8 Mb of the tetraploid genomes (from 0.4 Mb in S. spartinae to 7.9 Mb in S. versicolor) corresponding to ~ 0.04% to 1.16% of their genomes, respectively, but it is more abundant in the hexaploid species (i.e. 17 Mb in S. alterniflora and 35 Mb in S. maritima), corresponding to ~ 1% and 2% of their genomes, respectively. The relative abundance of this satellite in each hexaploid species was confirmed by Southern blot hybridization, which showed strong probe hybridization to S. maritima DNA and weaker hybridization to S. alterniflora DNA (Fig. 4c) reflecting sequence divergence, as supported by the detection of two independent clusters of reads using RepeatExplorer (Fig. 4d). The third satellite DNA sequence (with a monomer length of 370 bp) is the most abundant satellite in the hexaploid species S. alterniflora, representing up to 135 Mb (~7%) of its genome. In contrast, it was only detected in 0.15% of S. maritima reads. The lower abundance was also supported by the Southern blot hybridization (Fig. 4a) and FISH (Fig. 5) analyses. A comparative clustering analysis suggests that this satellite repeat is diverging between the two hexaploid species, given the observed spatial separation of the S. alterniflora and S. maritima sequences in the graphical depiction of three repeat clusters using Repeat Explorer (Fig. 4b). A comparison of the 370-bp satellite sequence in the NCBI database failed to find any evidence of homology between this satellite and centromeric repeats identified in other species.

Southern blotting (Fig. 4a) and FISH (Fig. 5) indicate that this 370 bp-long sequence is tandemly repeated and is physically located in subtelomeric positions on four chromosomes in S. maritima and on 42 chromosomes in S. alterniflora. It is notable that on two of the chromosomes in S. alterniflora the repeat was localized in the subtelomeric region of both chromosome arms (in contrast to all other chromosomes which had signal on just one of the chromosome arms). Cytogenetic analyses suggested that 14 chromosomes of the tetraploid S. spartinae also carried this repeat on one chromosome arm, even though this sequence was not detected in this J o u r n a l P r e -p r o o f species by in silico sequence analyses using RepeatExplorer. Semiquantitative PCR further revealed that the satellite sequence was also present (Supplementary Fig. S1), in low copy numbers in the tetraploids S. versicolor and S. bakeri and in high copy numbers in the hexaploid S. foliosa, as it occurs in S. alterniflora, its sister species.

Evolutionary dynamics of TEs

Phylogenetic analyses of the two main Gypsy lineages (Tekay and Ogre) detected in Spartina reveals contrasting evolutionary histories depending on species (Fig. 6a,b). Spartina versicolor and S. bakeri exhibit similar divergence profiles for Gypsy Tekay and Ogre elements, although some TE lineages were differentially enriched (Fig. 6a,b). For example, Tekay elements of lineage III (as determined from phylogenetic analyses of the RT domain, Fig. 6) are more abundant and divergent in S. bakeri ( S2).

In S. spartinae, pairwise sequence comparisons show higher divergence between reads coding for the reverse transcriptase (RT) domain of Tekay elements than in other tetraploid species (average distance of 0.41 in S. spartinae versus 0.22 and 0.35 in S. versicolor and S. bakeri, respectively; Fig. 6a), reflecting an older expansion of Tekay elements in this species. Spartina spartinae also displays the highest diversity of haplotypes: 803 haplotypes in Tekay lineage III and 417 haplotypes in lineage IV. This higher level of diversity in S. spartinae was typically found in each of the most abundant TE families identified (Fig. 6a-f, Supplementary Fig. S2). Interestingly, while all TEs were found to occur in intermediate quantities in S. spartinae compared with the other tetraploids and the two hexaploids, Ogre elements have accumulated in S. spartinae (104 Mb) compared with the other species (from 23 Mb to 74 Mb; Supplementary Table S2). In addition, elements from Ogre lineage III occupy a larger genome proportion in S. spartinae (68.7 Mb) than in other species (~10-14 Mb).

The two hexaploids, S. alterniflora and S. maritima, share similar divergence patterns for Gypsy Tekay elements, but these differ from the tetraploid species. Thus, the Tekay element profiles in hexaploids showed two distinct peaks, representing two predominant levels of divergence between RT domains: one with little average sequence divergence (0.13) and the other showing greater sequence divergence (0.51). These are likely to corresponds to two independent bursts during their common evolutionary history. In contrast, there is only a single peak of sequence divergence apparent in the tetraploids. The abundance and diversity of Tekay elements also differ between the two hexaploids species. For example, there is twice the amount of Tekay lineage IV derived reads in S. alterniflora (206 Mb) compared with S. maritima (106 Mb). In contrast, Tekay lineage III elements are more abundant in S. maritima (187 Mb) than in S. alterniflora (136Mb). There were no reads from S. alterniflora in Ogre lineages III and VI, whilst 64 haplotypes and 18 haplotypes were detected respectively of these lineages in S. maritima.

J o u r n a l P r e -p r o o f

Phylogenetic analysis of Copia SIRE elements (Fig. 6c) revealed three distinct lineages in all five Spartina species. The sister tetraploid species S. versicolor and S. bakeri show similar quantities and diversities for each lineage (lineage I: 14-16 Mb / 69-92 haplotypes; lineage II: 0.2-0.8 Mb / 5-7 haplotypes; lineage III: 7-8 Mb / 35-38 haplotypes). The hexaploid species S. alterniflora has approximately twice as many elements from lineage II (21 Mb) and more diversity (40 haplotypes) than S. maritima (10.8 Mb / 18 haplotypes), whereas S. maritima has about twice as many elements from lineage III (78 Mb) than S. alterniflora (35.8 Mb). For each SIRE lineage, S. spartinae showed an intermediate abundance of repeats compared with the other tetraploid and the hexaploid species (lineage I: 27 Mb; lineage II: 0.5 Mb; lineage III: 6 Mb), but this species had the greatest diversity of lineage I elements. The estimated divergence between reads showed a bimodal curve in all species with one peak closed to 0 (between 0 and 0.4) and another peak around 0.7. Compared to the patterns observed with Gypsy-derived sequences, most pairwise sequence comparisons in SIRE elements showed lower levels of divergence. This is also true for the Copia Ivana elements (Fig. 6d) where the curve decreases exponentially close to 0 (average distance between 0.05 and 0.09) in all species except S. spartinae, where reads tend to be slightly more divergent (average distance of 0.15).

The non-LTR LINE-RTE elements identified in Spartina species belong to a single lineage (Fig. 6e). As noted above for LTR-retrotransposons, the number of estimated haplotypes was not correlated with the abundance of this lineage in the investigated genomes. In the tetraploid species, the number of haplotypes varies from 31-37 in S. versicolor and S. bakeri to 135 in S. spartinae, whilst the amount of this repeat in the genome is typically around 3 Mb in all species. Amongst the hexaploids, S. alterniflora has a high level of diversity and a considerably larger amount of these repeats compared with tetraploids (129 haplotypes for 13 Mb). The repeat amount is even higher in S. maritima and yet the number of haplotypes is not as large as in S. alterniflora (38 haplotypes / 21 Mb). Divergence distance analyses confirm a remarkably low divergence between RT reads of LINE-RTE elements in all species.

Finally, six distinct lineages of CACTA elements were detected (Fig. 6f). All lineages contain sequences from all five species with the exception of lineage IV which does not appear to include sequences from S. alterniflora. Each lineage is similar in diversity and quantity in the two tetraploids S. versicolor and S. bakeri.

In contrast, S. spartinae has a higher diversity of elements in all lineages than the two tetraploid species. Amongst the hexaploids, S. maritima contains more CACTA elements in each lineage than S. alterniflora (lineage I to V). Pairwise sequence comparisons in CACTA elements showed the same low divergence in all species with only one peak around 0.1-0.2.

Phylogenetic and divergence analyses of the other identified TE-families are available in Supplementary Fig. S3.

Discussion

Repeat content in Spartina and comparisons with other genomes in Poaceae

By comparing genomic sequences in related tetraploid and hexaploid Spartina species, we shed light on the evolutionary dynamics of the repetitive sequence component comprising their genomes over the last 6-10 million years (estimated time since Spartina diverged from its last common ancestor in subfamily Chloridoideae [START_REF] Rousseau-Gueutin | The chloroplast genome of the hexaploid Spartina maritima (Poaceae, Chloridoideae): comparative analyses and molecular dating[END_REF]). The genome sizes of tetraploids are smaller (1.39-1.97 pg/2C, 8 species) than hexaploids (3.67 -4.05 pg/2C, 3 species). This difference is not only accounted for by the higher ploidy levels, since dividing the J o u r n a l P r e -p r o o f genome size by ploidy level reveals that the monoploid genome sizes (1Cx-value; [START_REF] Greilhuber | Intraspecific variation in genome size in Angiosperms: identifying its existence[END_REF]) of the tetraploid species are also considerably smaller (0.34-0.49 pg/1Cx) than those of the hexaploid species (0.61-0.68 pg/1Cx) (Table 1). Thus, there is likely to have been genome upsizing in the hexaploids and/or downsizing in the tetraploids associated with species radiations.

Spartina belongs to subfamily Chloridoideae, one of the poorest investigated lineages in the grass family.

Our analyses have allowed us to identify the repeat fraction of the genomes in five Spartina species. These genomes are only ~ 33% -45% repetitive depending on species, which indicates limited amplification of repeats, or more efficient removal of repeats, compared with other grasses, such as Zea mays, Hordeum vulgare and Triticum aestivum which are estimated to be ~ 80% repetitive [10,[START_REF] Wicker | Impact of transposable elements on genome structure and evolution in bread wheat[END_REF][START_REF] Mascher | A chromosome conformation capture ordered sequence of the barley genome[END_REF]). The estimated abundances of repeats in Spartina are similar to those reported for the allotetraploid Eragrostis tef (27%) which belongs to the same subfamily as Spartina (i.e. Chloridoideae) and whose genome size of 730 Mb/1C [START_REF] Ayele | Flow cytometric analysis of nuclear genome of the Ethiopian cereal tef [Eragrostis tef (Zucc.) Trotter[END_REF][START_REF] Gebre | Identification and characterization of abundant repetitive sequences in Eragrostis tef cv. Enatite genome[END_REF] is broadly similar to the tetraploid species S. versicolor and S. bakeri. Potentially older more degraded TEs occur in the genomes of Spartina, but they remain undetected using the homology parameters used here (80% of min. identity).

The majority of repeated sequences are LTR-retrotransposons representing between 24% and 38% of the genome depending on Spartina species (Table 3). Amongst LTR-retrotransposons, Gypsy elements are more abundant (17% to 30%) than Copia elements (from 6% to 10%). This is commonly observed in Poaceae, for example, Zea mays (76% of Gypsy vs. 9% of Copia), Sorghum bicolor (19% vs. 5%) and Setaria italica (22% vs. 7%) [10,[START_REF] Paterson | The Sorghum bicolor genome and the diversification of grasses[END_REF]109]. The non-LTR retrotransposon LINE elements and DNA transposons represent only a small fraction of Spartina genomes (less than 1% of LINEs and from 1.5% to 4.4% of DNA transposons) as is also typically reported for many plant genomes analyzed so far [START_REF] Kejnovsky | Plant transposable elements: biology and evolution[END_REF].

Within Spartina, different repeat families evolved through differential expansion or deletion in the tetraploid and hexaploid lineages. Thus, each investigated species is now characterized by a distinctive repeat profile, with the most closely related species showing the most similar profiles. For several repeat elements, pairwise sequence comparisons revealed a bimodal profile of sequence similarities (Fig. 6). This could represent two independent bursts of repeat activity during their evolution within the genomes of different species -one wave of activity that is still ongoing giving rise to a population of repeats which have high levels of sequence similarity, and another more ancient burst of repeat activity generating a peak of more degenerate copies.

Evolutionary dynamics of TEs in tetraploid Spartina species

The two closely related tetraploids species S. versicolor and S. bakeri are sister taxa which are thought to have diverged less than 2 mya [START_REF] Rousseau-Gueutin | The chloroplast genome of the hexaploid Spartina maritima (Poaceae, Chloridoideae): comparative analyses and molecular dating[END_REF] and have similar genome sizes (680 Mb/1C and 689 Mb/1C, respectively).

Their genomes contain 33% of repeats (~230 Mb) comprising approximately 17% Gypsy, 6% Copia, 3-4% DNA transposons and 4-5% of satellite repeats (Table 3, Fig. 2,3,6). Similar genome sizes and repeat profiles indicate no major changes in TE content since their divergence from their common ancestor ~ 2 mya. In contrast, the tetraploid S. spartinae, whose phylogenetic relationship to other Spartina species is still debated [START_REF] Baumel | Molecular phylogeny of hybridizing species from the genus Spartina Schreb. (Poaceae)[END_REF][START_REF] Rousseau-Gueutin | The chloroplast genome of the hexaploid Spartina maritima (Poaceae, Chloridoideae): comparative analyses and molecular dating[END_REF], exhibited more divergent features of its repeat landscape, which comprises 44% of its genome (~421 Mb). Not only does this species have the largest tetraploid genome analyzed (1C = 963 Mb) with about two-fold more repetitive DNA than the other analyzed tetraploids, but the diversity of repeats in terms of the number of J o u r n a l P r e -p r o o f haplotypes in each family is also high, resembling more closely that seen for the hexaploids. For example, the Ogre lineage III constituted 7% of the S. spartinae genome (i.e. 69 Mb) but represented only a limited fraction of the S. versicolor (1.5%) and S. bakeri (2.1%) genomes (i.e. 10 and 14 Mb respectively). In addition, the number of Ogre lineage III haplotypes is greater in S. spartinae (294 haplotypes vs 158 and 127 haplotypes in S. versicolor and S. bakeri respectively).

Biological traits such as mode of reproduction, the degree of selfing versus outcrossing have all been suggested to impact TE dynamics [START_REF] Ågren | Evolutionary transitions in individuality: insights from transposable elements[END_REF]. Spartina species are all perennial, wind pollinated plants. Asexual reproduction is significant for the propagation and invasive success of S. versicolor (syn. S. patens) in introduced European populations, where seed production is scarce [START_REF] Baumel | Spartina versicolor Fabre: another case of Spartina trans-Atlantic introduction?[END_REF][START_REF] Sánchez | Can asexual reproduction by plant fragments help to understand the invasion of the NW Iberian coast by Spartina patens?[END_REF]. However, little is known about the relative importance of sexual versus asexual reproduction in the reproductive biology of S. bakeri and S. spartinae.

The diversity and quantity of repeats in S. spartinae suggests different patterns of repeat proliferation and divergence compared with the other tetraploids, or the early divergence of this species from the other tetraploids.

However, the precise phylogenetic placement of S. spartinae is unresolved from phylogenetic studies published so far [START_REF] Baumel | Molecular phylogeny of hybridizing species from the genus Spartina Schreb. (Poaceae)[END_REF][START_REF] Fortune | Evolutionary dynamics of Waxy and the origin of hexaploid Spartina species (Poaceae)[END_REF][START_REF] Rousseau-Gueutin | The chloroplast genome of the hexaploid Spartina maritima (Poaceae, Chloridoideae): comparative analyses and molecular dating[END_REF], with some evidence suggesting that it may have diverged early in Spartina, while other data indicating that it is sister to the hexaploid clade (Fig. 1). Our data might support the latter assertion, but it will be necessary to explore the TE content of other tetraploid Spartina species (Fig. 1), such as S. arundinaceae (1C = 782 Mb) and S. pectinata (1C = 763 Mb), to better resolve the patterns of gains and/or losses of repeats from their common ancestor. Given the uncertain phylogenetic position of S. spartinae, several alternative hypotheses about the past dynamics of TEs could explain the repeat patterns observed in S. spartinae. (i) The high TE divergence in S. spartinae could reflect ongoing mutation accumulation of old repeat elements inherited from the ancestral genome of Spartina. The lower amount and diversity of TEs observed in S. versicolor and S. bakeri would thus reflect a loss of these elements associated with genome downsizing from their ancestor.

Studies have shown that in some cases genome downsizing can occur rapidly and drastically following polyploidy [START_REF] Leitch | Genomic plasticity and the diversity of polyploid plants[END_REF][START_REF] Renny-Byfield | Next generation sequencing reveals genome downsizing in allotetraploid Nicotiana tabacum, predominantly through the elimination of paternally derived repetitive DNAs[END_REF][START_REF] Renny-Byfield | Independent, rapid and targeted loss of highly repetitive DNA in natural and synthetic allopolyploids of Nicotiana tabacum[END_REF][START_REF] Koukalova | Fall and rise of satellite repeats in allopolyploids of Nicotiana over c. 5 million years[END_REF][START_REF] Leitch | The ups and downs of genome size evolution in polyploid species of Nicotiana (Solanaceae)[END_REF], but it has also been shown to occur more progressively in other species [START_REF] Renny-Byfield | Doubling down on genomes: polyploidy and crop plants[END_REF][START_REF] Wendel | The long and short of doubling down: polyploidy, epigenetics, and the temporal dynamics of genome fractionation[END_REF], as may be apparent amongst some of the tetraploid Spartina species. (ii) The high diversity of repeats observed in S. spartinae could be explained by a hybrid origin of this species. This hypothesis could be consistent with the conflicting phylogenetic signal over its placement. Considering its current geographic range, S. spartinae has a disjunct distribution in South America (northern Argentina and Paraguay) and North America (Gulf coast in USA, and Mexico to Costa Rica) where several Spartina species such as S. alterniflora, S. patens, S. bakeri and S. cynosuroides are encountered [START_REF] Marchant | Evolution in Spartina (Gramineae): III. Species chromosome numbers and their taxonomic significance[END_REF][START_REF] Mobberley | Taxonomy and distribution of the genus Spartina[END_REF]. A complex reticulate history of S. spartinae may therefore have occurred, involving, for example, introgression and/or horizontal gene transfers [START_REF] Baidouri | Widespread and frequent horizontal transfers of transposable elements in plants[END_REF][START_REF] Peccoud | Analyzing horizontal transfer of transposable elements on a large scale: challenges and prospects[END_REF]. It is therefore possible that conflicts between TEs of multiple evolutionary origins and their repressors could have contributed to increasing the expansion of some TE lineages. Such processes could certainly have given rise to a complex repeat profile as a result of repeats being inherited from two or more species. (iii) A non-exclusive likely hypothesis is that current TEs in S. spartinae reflect TE accumulation following bursts of activity in the common ancestor of S. spartinae and the hexaploid species, which also have a greater diversity of repeats than the other tetraploids. Such arguments would reinforce the hypothesis of a close phylogenetic relationship between the hexaploids and S. spartinae, which would then represent one of the tetraploid ancestor lineages involved in the formation of the hexaploid Spartina clade.

J o u r n a l P r e -p r o o f

Differential evolution of TEs in hexaploid Spartina species

The hexaploid species S. alterniflora and S. maritima diverged around 2-4 Mya [START_REF] Rousseau-Gueutin | The chloroplast genome of the hexaploid Spartina maritima (Poaceae, Chloridoideae): comparative analyses and molecular dating[END_REF] on the American and Euro-African Atlantic coasts, respectively, and they display distinct repeat profiles. Both species have a similar fraction of their genome being repetitive (i.e. 45.5%, three-fold more than previously reported from BAC-end sequencing [START_REF] Ferreira De Carvalho | Exploring the genome of the salt-marsh Spartina maritima (Poaceae, Chloridoideae) through BAC end sequence analysis[END_REF]). However, there is a greater quantity of TEs in S. maritima (estimated to 751 Mb) than in S. alterniflora (724 Mb) and a greater quantity of satellite DNAs in S. alterniflora (164 Mb) than in S. maritima (66 Mb). This is consistent with previous transposon display analyses [START_REF] Parisod | Rapid structural and epigenetic reorganization near transposable elements in hybrid and allopolyploid genomes in Spartina[END_REF]) who suggested greater accumulation of TEs in S. maritima than S. alterniflora.

Within TEs, Tekay and CRM lineages were more abundant in S. alterniflora while Ogre and SIRE lineages were more abundant in S. maritima (Fig. 2 and Supplementary Table S1). All Gypsy, SIRE and CACTA lineages (representing the majority of TEs) displayed high diversity (i.e. many haplotypes) with similar divergence patterns in the two species (Fig. 6) suggesting that the TEs in S. maritima and S. alterniflora were derived from the same transposition bursts before they diverged 2-4 mya but have since evolved independently.

However, the number of detected copies in the Copia Ivana and LINE-RTE (non-LTR retrotransposon) lineages was lower in S. maritima than S. alterniflora. Pairwise sequence comparisons suggest in addition, high sequence similarity between many of the S. maritima reads indicative of recent bursts of amplification in this species. Similar results were observed for Copia Ikeros in S. alterniflora (Supplementary Fig. S3). A recent study of the evolution of ra-siRNAs (repeat-associated small-interfering RNA) in S. alterniflora and S. maritima identified many ra-siRNAs targeting preferentially Ivana, Ikeros and LINE-RTE lineages [START_REF] Cavé-Radet | Evolution of small RNA expression following hybridization and allopolyploidization: insights from Spartina species (Poaceae, Chloridoideae)[END_REF]. Such results are consistent with the hypothesis that these elements (i.e. Ivana and LINE-RTE elements in S. maritima and Ikeros elements in S. alterniflora) have recently been and/or are still active, with their activity being regulated via lineage-specific siRNAs. Further work is needed to determine if the elements are transcriptionally active and if they are able to insert into the genome.

Expansion of satellite sequences in S. alterniflora

Three different satellite DNA sequences were identified in Spartina that range in abundance from 1.6% (15.6 Mb) in the genome of S. spartinae to 8.4% (164.3 Mb) in S. alterniflora. Two of them (a 167 bp-long monomer and a 147 bp-long monomer) were found to be similar to centromeric repeats previously characterized in grasses, including in Zea mays, Oryza, Sorghum and Brachypodium [START_REF] Aragón-Alcaide | A cereal centromeric sequence[END_REF][START_REF] Jiang | A conserved repetitive DNA element located in the centromeres of cereal chromosomes[END_REF][START_REF] Ananiev | Chromosome-specific molecular organization of maize (Zea mays L.) centromeric regions[END_REF][START_REF] Dong | Rice (Oryza sativa) centromeric regions consist of complex DNA[END_REF]. The 147 bp monomer repeat was more abundant in the two closely related tetraploid Spartina species (S. versicolor and S. bakeri), while the 167 bp monomer sequence was more abundant in the hexaploid species (validated by Southern blotting, Fig. 4). Out of the three tetraploids, the genome of S. spartinae had the smaller fraction of satellite DNA overall (only 1.6% vs. 4-5% in the other tetraploids; Table 3), highlighting that in contrast to the TEs discussed above, these satellite repeats were not contributing to its larger genome size.

A third remarkable satellite sequence with a monomer size of 370 bp was found to be especially abundant in the hexaploid S. alterniflora, where it represents ~7% of the analyzed genomic sequences. This agrees with previous cytogenetic observations [START_REF] Renny-Byfield | Flow cytometry and GISH reveal mixed ploidy populations and Spartina nonaploids with genomes of S. alterniflora and S. maritima origin[END_REF] suggesting an abundance of heterochromatin on S. alterniflora chromosomes. No sequence similarity to other known satellite DNAs was observed, even to satellite sequences reported in a related Chloridoideae species Eragrostis tef [START_REF] Gebre | Identification and characterization of abundant repetitive sequences in Eragrostis tef cv. Enatite genome[END_REF]. Southern blotting allowed us to confirm the J o u r n a l P r e -p r o o f presence of this 370-bp tandem repeat to be in higher abundance in S. alterniflora (135 Mb) than S. maritima (~3 Mb). The sequence was localized by FISH to subtelomeric positions on 42 chromosomes of S. alterniflora (2n = 62), and to just four chromosomes in S. maritima (2n = 60) (Fig. 4). Interestingly, this repeat was mostly localized to the subtelomeric region of just one of the two chromosome arms in 40 out of 42 chromosomes of S. alterniflora and the four chromosomes of S. maritima. The remaining two chromosomes of S. alterniflora with FISH signal showed the 370-bp satellite was localized in the subtelomeric regions of both chromosome arms. These chromosomes might represent a fixed pair of supernumerary or B chromosomes in S. alterniflora, whose karyotype is 2n = 60+2 compared with S. maritima which is 2n = 60 [START_REF] Marchant | Evolution in Spartina (Gramineae): III. Species chromosome numbers and their taxonomic significance[END_REF]. These two extra chromosomes are inherited together in hybrids involving S. alterniflora (e.g. S.  townsendii (2n = 60+2) [START_REF] Groves | Spartina townsendii[END_REF] and S.  neyrautii (2n = 60+2) [125]). Potentially this satellite repeat has a key function in chromosome segregation during meiosis, "driving" the supernumerary chromosomes into the next generation, as found for similar satellite DNA sequences on B or supernumerary chromosomes in other species [START_REF] Garrido-Ramos | Satellite DNA in plants: more than just rubbish[END_REF][START_REF] Garrido-Ramos | Satellite DNA: an evolving topic[END_REF].

Semi-quantitative PCR indicated this 370-bp satellite was also present in the hexaploid S. foliosa (2n = 62; 2C = 4.05pg) in similar abundance to S. alterniflora (Supplementary Fig. S1). This is consistent with the phylogenetic relationships between these two species as they are considered to be sister species within the hexaploid Spartina subclade (Fig. 2) [START_REF] Baumel | Molecular phylogeny of hybridizing species from the genus Spartina Schreb. (Poaceae)[END_REF][START_REF] Fortune | Evolutionary dynamics of Waxy and the origin of hexaploid Spartina species (Poaceae)[END_REF][START_REF] Rousseau-Gueutin | The chloroplast genome of the hexaploid Spartina maritima (Poaceae, Chloridoideae): comparative analyses and molecular dating[END_REF]. Thus, it is likely that the evolution of this satellite DNA occurred prior to the divergence of these species. If the sequence shows 'meiotic drive' as the results from S. alterniflora suggest, its occurrence more widely amongst Spartina species may be important for the evolution of linked alleles.

The 370-bp satellite was not detected in silico in the genomic sequence datasets from the tetraploid species.

Nevertheless, FISH and semi-quantitative PCR analyses indicated it was present in low amounts in S. versicolor and S. bakeri, and on 14 chromosomes in S. spartinae (Fig. 5). These data suggest that this satellite may have been present in the common ancestor of Spartina. Various studies have shown that satellite DNA sequences can evolve rapidly and their amount can vary considerably, even between closely related species [START_REF] Mehrotra | Repetitive sequences in plant nuclear DNA: types, distribution, evolution and function[END_REF][START_REF] Renny-Byfield | Independent, rapid and targeted loss of highly repetitive DNA in natural and synthetic allopolyploids of Nicotiana tabacum[END_REF][START_REF] Garrido-Ramos | Satellite DNA: an evolving topic[END_REF][START_REF] Plohl | Satellite DNAs between selfishness and functionality: Structure, genomics and evolution of tandem repeats in centromeric (hetero)chromatin[END_REF][START_REF] Plohl | Satellite DNA evolution[END_REF].

In conclusion, our comparative analyses of five polyploid species representative of the main Spartina clades, reveal contrasting TE dynamics and satellite DNA accumulation within both tetraploid and hexaploid lineages. Repetitive sequence dynamics reflects the long-term evolution associated with allopatric speciation between hexaploids that diverged 3-6 MYA between the American (S. alterniflora) and European (S. maritima) Atlantic coasts respectively, or between North-American (S. bakeri S. versicolor sub-clade) and South-American (S spartinae) tetraploids, in agreement with their phylogenetic history. Genome expansion or contraction resulting from repeat evolution seems to be mostly determined by the divergence times between species, rather than by genomic changes triggered by ploidy level change per se, as suggested by our earlier studies on Spartina neopolyploids, where the immediate response to allopolyploidy affects epigenetic regulation of repeats.

The results obtained here, which include repeat content evaluation and characterization between Spartina lineages, provide crucial insights for future investigations into the genetic, epigenetic and transcriptomic consequences of differential repeat dynamics in the meso-and neopolyploid species.

J o u r

  n a l P r e -p r o o f Chromovirus (Tekay and CRM) and Ikeros lineages are more abundant in S. alterniflora (estimated to 399 Mb and 25 Mb, respectively) than in S. maritima (314 Mb and 9 Mb). In contrast, Ogre and SIRE lineages are more abundant in S. maritima (74 Mb and 143 Mb respectively) than in S. alterniflora (37 Mb and 126 Mb).

  The detection of monomers (only in S. maritima), dimers and trimers in Southern blots (identification of band sizes corresponding to 167-bp repeats) also demonstrate the tandem arrangement of units. A second satellite repeat (monomer-length of 147 bp) is more abundant in S. versicolor (18 Mb) and S. bakeri (17 Mb) than in S. spartinae (6 Mb) and both hexaploids (1.3 and 10 Mb in S. alterniflora and S. maritima, respectively). These two satellite DNAs have sequence homology to SAT1 and CentC (genbank ID: M32521.1 and KT724912.1) which are two centromeric repeats identified in several grasses like rice, maize, Sorghum and Brachypodium[START_REF] Dennis | Knob heterochromatin homology in maize and its relatives[END_REF][START_REF] Aragón-Alcaide | A cereal centromeric sequence[END_REF][START_REF] Jiang | A conserved repetitive DNA element located in the centromeres of cereal chromosomes[END_REF][START_REF] Ananiev | Chromosome-specific molecular organization of maize (Zea mays L.) centromeric regions[END_REF][START_REF] Dong | Rice (Oryza sativa) centromeric regions consist of complex DNA[END_REF].

J o u r 2 J

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Fig. 2

 2 Fig. 2 Proportion of TEs and satellite sequences in the genomes of the five studied Spartina species. Tetraploid species are coloured in green and hexaploid species in blue.

Fig. 3

 3 Fig.3Amount of satellite sequences found in hexaploid and tetraploid Spartina species. Three different satellite repeats were detected, two of which (i.e. 147bp and 167 bp repeats) showed sequence homology with centromeric repeats of grasses. The third satellite repeat of 370bp was abundant in S. alterniflora, where it represents approximately 7% of the genome.

  r n a l P r e -p r o o f

Fig. 4

 4 Fig. 4 Southern blot hybridization of genomic DNA from S. alterniflora and S. maritima with the 370-bp satellite (A) and the 167-bp satellite (C). (A) Regularly spaced hybridization signals in S. alterniflora show that the 370-bp satellite was abundant and tandemly repeated in this species. In contrast, in S. maritima has only a weak signal (see arrow) which confirms its presence, but in limited quantity. (B) Comparative graph genomic analysis of the 370-bp satellite (partitioned in 3 clusters). Nodes symbolize reads (green for S. alterniflora and blue for S. maritima) and the sizes of edges are proportional to the divergence between two related reads. Reads from 370-bp satellite exhibit a relatively low divergence in S. alterniflora (tightly clustered reads) compared with S. maritima, indicating a burst of divergence in the latter. (C) Southern blot hybridization confirms that the 167-bp satellite is more abundant in S. maritima genome than in S. alterniflora. (D) Graphical analyses indicate a genome specific diversity of the 167-bp satellite (each cluster specific to one species).

  r n a l P r e -p r o o f

Fig. 6

 6 Fig. 6 Phylogenetic analyses and pairwise sequence comparisons (Kimura 2-parameters distance) of the main TEs found in Spartina species based on reverse transcriptase coding sequences for Class I elements (a-e) or transposase coding sequences for Class II CACTA elements (f). According to phylogenetic relationships between sequences, TEs were classified into different monophyletic lineages (coded in roman numbers). The estimated diversity (number of detected haplotypes) and estimates of their genome contribution (in Mb) are indicated for each lineage. The sequences are color coded to species as shown in (b).

  54.2 Mb; 177 haplotypes) than in S. versicolor (35 Mb; 68 haplotypes). Conversely, Tekay elements in lineage IV occurred in greater quantity but lower sequence diversity in S. versicolor (37.7 Mb; 36 haplotypes) than in S. bakeri (21.8 Mb; 71). Similar differential accumulation of TEs between tetraploid species is also observed for Gypsy Ogre elements (Fig. 6b). Despite these differences, both tetraploid species contain similar overall amounts of Tekay elements (72.8 Mb and 76.3 Mb in S. versicolor and S. bakeri, respectively) and Ogre elements (37.3 Mb and 36.3 Mb, respectively)

(Supplementary Table

Table 3

 3 Annotation and estimated amounts of major TE lineages found in the five investigated Spartina species

			S. alterniflora			S. maritima			S. spartinae		S. versicolor		S. bakeri
			1C = 1,956 Mb			1C = 1,795 Mb			1C = 963 Mb		1C = 680 Mb		1C = 689 Mb
	Order	Superfamily	# cluster	Estimated quantity in the genome (Mb)	% genome	of	# cluster	Estimated quantity in the genome (Mb)	% genome	of	# cluster	Estimated quantity in the genome (Mb)	% of genome # cluster	Estimated quantity in the genome (Mb)	% of genome # cluster	Estimated quantity in the genome (Mb)	% genome	of
	Class I Retrotransposon	39	693.5	35.48% 38	681.8	37.99% 37	367.5	38.17%	30	165.8	24.37%	34	172.0	24.55%
		Gypsy	19	491.0	25.11% 23	473.4	26.38% 15	294.9	30.62%	10	117.2	17.22%	15	124.0	17.58%
	LTR	Copia	15	186.2	9.53%		12	185.1	10.32% 18	67.0	6.97%	14	40.0	5.89%	14	40.5	5.89%
		Other LTR	2	1.7	0.09%		1	1.8	0.10%		1	1.2	0.13%	2	3.8	0.56%	2	3.1	0.45%
	LINE-RTE		1	13.6	0.7%		1	20.7	1.15%		1	3.7	0.38%	1	3.1	0.45%	1	2.9	0.42%
	SINE		2	1.0	0.05%		1	0.8	0.04%		2	0.7	0.07%	3	1.7	0.25%	2	1.5	0.21%
	Class II DNA-transposons	8	30.3	1.55%		10	69.4	3.87%		21	38.0	3.95%	13	23.4	3.45%	13	30.6	4.43%
	TIR	CACTA Other TIR	6 1	26.0 0.4	1.33% 0.02%		5 5	50.7 10.2	2.82% 0.57%		9 10	23.5 14.0	2.44% 1.43%	6 6	18.9 4.3	2.78% 0.64%	6 6	18.0 12.4	2.61% 1.79%
	Helitron		1	3.9	0.2%		1	8.5	0.48%		2	0.8	0.08%	1	0.2	0.03%	1	0.2	0.03%
	Tandem repeats / satellite	20	164.3	8.40%		11	66.4	3.71%		14	15.6	1.62%	20	34.7	5.10%	27	27.7	4.01%
	TOTAL Repeat DNA	67	888.1	45.43% 60	817.6	45.57% 72	421.1	43.74%	63	223.9	32.92%	74	230.3	33.40%

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Acknowledgements

We thank Dr. A. Baumel (IMBE, Aix-Marseille University, France), Dr. T. Columbus (Rancho Santa Ana Botanical Garden, CA USA) for their help in getting Spartjna plant material, as well as T. Fontaine and R. Bodiguel (Experimental Ecology platform, UMR CNRS Ecobio, University of Rennes 1, France) for maintaining the Spartina living collection in the Greenhouse. M. Bourges and N. Valentin (Institute of Integrative Biology of the Cell, Gif s/Yvette) are thanked for flow cytometry analyses. We acknowledge the perspicacity of late Dr. K.Y. Lim (Queen Mary University of London) who early suggested from cytogenetic observations the abundance of satellite repeats in S. alterniflora.

Funding This work was financed under the International Associated Laboratory "Ecological Genomics of Polyploidy" supported by CNRS (INEE, UMR CNRS 6553 Ecobio), University of Rennes 1 and the Iowa State University (USA). The analyses benefited from the Molecular Ecology (UMR CNRS 6553 Ecobio) and Genouest (Biogenouest) facilities. Delphine Giraud benefited from a PhD scholarship from the University of Rennes 1.

Conflicts of interest

The authors declare that they have no conflict of interest

Authors' contributions