Yohan Pillon

Pauline Majourau

Karine Gotty

Sandrine Isnard

Bruno Fogliani

Mark W Chase

Gael J Kergoat

The allopolyploid origin(s) and diversification of New Caledonian Grevillea (Proteaceae)

Keywords: Adaptive radiation, genome duplication, island biogeography, manganese, seeds, serpentine ORCID

The assembly of island plant communities is the result of a number of processes: immigration (dispersal), speciation, and extinction. Using four plastid genes and one lowcopy nuclear gene, we investigated the origin of the New Caledonian Grevillea (Proteaceae), an otherwise largely Australian genus. In the combined plastid analysis, the species form two distinct clades, the exul (four species) and gillivrayi groups (six species), within group 3 of Grevillea. All New Caledonian Grevillea display two distinct copies of PHYA, one copy in group 3, and another in group 4. Previously published chromosome counts for G. meisneri and two new genome size estimates of G. gillivrayi and G. rubiginosa suggest that these plants are tetraploids. Altogether, the current data available suggest that New Caledonian Grevillea are allotetraploids, resulting from one or two hybridisation events between two or three distinct parents. A possible scenario is that modern Grevillea has descended from a hybrid swarm that formed 9-13 Mya between multiple immigrants (now extinct) that reached the island. Grevillea can be added to the list of island plant radiations with an early history of hybridisation and polyploidy. The relative importance, location and timing of these two mechanisms remain to be elucidated. The two groups of New Caledonian Grevillea may be easily distinguished by their inflorescences, flowers and fruits, with the gillivrayi group displaying greater ecological and morphological diversity. The seed characteristics of New Caledonian Grevillea are consistent with their pioneer behavior.

Introduction

Islands are widely recognised as unparalled evolutionary laboratories, where the genesis of biodiversity is expected to be easier to investigate. Two central themes in island biology are the importance of arrival history in community assembly and the genomic signature of adaptation and speciation [START_REF] Warren | Islands as model systems in ecology and evolution: prospects fifty years after MacArthur-Wilson[END_REF]. The most typical examples of adaptive radiation in island plants include Hawaiian silverswords (Asteraceae) and Hawaiian lobeliads (Campanulaceae), which have in common polyploidy. In the case of the silverswords, the allopolyploidisation event shortly pre-dated their radiation [START_REF] Barrier | Interspecific hybrid ancestry of a plant adaptive radiation: allopolyploidy of the Hawaiian silversword alliance (Asteraceae) inferred from floral homeotic gene duplications[END_REF]). Although it is accepted that Hawaiian lobeliads are all tetraploid [START_REF] Lammers | Chromosome numbers and their systematic implications in Hawaiian Lobelioideae (Campanulaceae)[END_REF], it is not clear whether they are auto-or allotetraploids and when the genome duplication event occurred, this being a recurrent trend in Lobelioideae [START_REF] Antonelli | Higher levels phylogeny and evolutionary trends in Campanulaceae subfam. Lobeloideae: Molecular signal overshadows morphology[END_REF]. In another Hawaiian plant radiation, Cyrtandra (Gesneriaceae), there is evidence that the island of Hawai'i was colonized by multiples lineages that had uneven success and hybridized [START_REF] Johnson | Multiple colonizations, hybridization and uneven diversification in Cyrtandra (Gesneriaceae) lineages on Hawai'i Island[END_REF]. Although multiple colonisations, hybridization and polyploidisation may be three important features in the genesis of island plant diversity, their frequency and role may have been underestimated.

It is increasingly accepted that long-distance dispersal is more frequent than was previously thought (de Queiroz 2005; Christenhusz and Chase 2013). Long-distance dispersal through wind, birds and ocean currents is likely the major process involved in patterns of plant disjunct distributions [START_REF] Gillespie | Long-distance dispersal: a framework for hypothesis testing[END_REF], in particular in the case of some a priori unlikely colonisation routes. For example, Acridocarpus (Malpighiaceae) reached New Caledonia from Madagascar [START_REF] Davis | Phylogeny of Acridocarpus-Brachylophon (Malpighiaceae): implications for tertiary tropical floras and afroasian biogeography[END_REF], Chamaedoreeae (Arecaceae) and Weinmannia (Cunoniaceae) reached the Mascarenes from the Americas [START_REF] Baker | Global biogeography and diversification of palms sheds light on the evolution of tropical lineages. I. Historical biogeography[END_REF][START_REF] Pillon | Phylogenomics and biogeography of Cunoniaceae (Oxalidales) with complete generic sampling and taxonomic realignments[END_REF]), and Acacia s.s. (Fabaceae) reached Réunion from the Hawaiian Islands (Le [START_REF] Roux | Relatedness defies biogeography: the tale of two island endemics (Acacia heterophylla and A. koa)[END_REF]. In spite of being the most isolated archipelagos on Earth, the Hawaiian Islands have been colonized multiple times by several plant genera:

Coprosma (Rubiaceae) [START_REF] Cantley | Biogeographic insights on Pacific Coprosma (Rubiaceae) indicate two colonizations to the Hawaiian Islands[END_REF], Santalum (Santalaceae) [START_REF] Harbaugh | Phylogeny and biogeography of the sandalwoods (Santalum, Santalaceae): repeated dispersal throughout the Pacific[END_REF], and Scaevola (Goodeniaceae) [START_REF] Howarth | Phylogenetics of the genus Scaevola (Goodeniaceae): implication for dispersal patterns across the Pacific basin and colonization of the Hawaiian islands[END_REF]. At least eight dispersals of Hawaiian plants to the distant Marquesas have also been reported [START_REF] Price | Origins of the Hawaiian flora: Phylogenies and biogeography reveal patterns of long-distance dispersal[END_REF].

Hybridization is an important process on islands, although "reports of hybridization in insular floras depend on the tendency of systematists to recognize the presence of hybrids or to neglect them" (Carlquist 1974 p. 534). It therefore varies according to archipelago and taxa [START_REF] Stuessy | Interpretation of patterns of genetic variation in endemic plant species of oceanic islands[END_REF]. For example, hybridization is generally recognised as a common feature in the Hawaiian flora, e.g. Cyrtandra [START_REF] Smith | Interspecific hybridization in natural populations of Cyrtandra (Gesneriaceae) on the Hawaiian islands : evidence from RAPD[END_REF], Metrosideros (Myrtaceae) [START_REF] Stacy | Genetic analysis of an ephemeral intraspecific hybrid zone in the hypervariable tree, Metrosideros polymorpha, on Hawai'i Island[END_REF], Santalum [START_REF] Harbaugh | Polyploid and hybrid origins of pacific island sandalwoods (Santalum, Santalaceae) inferred from low-copy nuclear and flow cytometry data[END_REF], and Scaevola [START_REF] Howarth | Genealogical evidence of homoploid hybrid speciation in an adaptive radiation of Scaevola (Goodeniaceae) in the Hawaiian islands[END_REF] and also potentially important in the Macaronesian flora [START_REF] Herben | The ghost of hybridization past: niche pre-emption is not the only explanation of apparent monophyly in island endemics[END_REF]). In the flora of New Caledonia, evidence is scarcer, with some reports (mostly recent) in Araucaria (Araucariaceae) [START_REF] Gaudeul | Evolutionary dynamics of emblematic Araucaria species (Araucariaceae) in New Caledonia: nuclear and chloroplast markers suggest recent diversification, introgression, and a tight link between genetics and geography within species[END_REF], Corybas (Orchidaceae) [START_REF] Faria | Diversité du genre Corybas Salisb. (Orchidaceae, Diurideae) en Nouvelle-Calédonie[END_REF], Dacrydium (Podocarpaceae) [START_REF] Knopf | Dacrydium × suprinii , sp. nova -a natural hybrid of Dacrydium araucarioides × D. guillauminii[END_REF]) and multiple genera of Cunoniaceae [START_REF] Pillon | Two new species of Cunonia (Cunoniaceae) from New Caledonia[END_REF]Pillon, Munzinger, et al. 2009;Pillon, Hopkins, et al. 2009). Hybridisation may be advantageous to colonise novel habitats [START_REF] Rieseberg | Hybridization and the colonization of novel habitats by annual sunflowers[END_REF] or promote invasiveness [START_REF] Schierenbeck | Hybridization and the evolution of invasiveness in plants and other organisms[END_REF].

Polyploidy may be an important feature of island floras. For instance, polyploids may represent as much as 80% of the native Hawaiian flora [START_REF] Carr | Chromosome evolution and speciation in Hawaiian flowering plants[END_REF]. Polyploidy is also known as an important driver of diversification, as in the case of Allium (Amaryllidaceae) [START_REF] Han | Polyploidy promotes species diversification of Allium through ecological shifts[END_REF]. It may facilitate the invasion of introduced plants [START_REF] Ainouche | Hybridization, polyploidy and invasion: lessons from Spartina (Poaceae)[END_REF][START_REF] Te Beest | The more the better? The role of polyploidy in facilitating plant invasions[END_REF], thereby potentially helping colonisation and settlement on islands. However information on chromosome numbers is uneven between islands, e.g. Hawai'i (50 % with chromosome counts) and New Zealand (86%) [START_REF] Meudt | Polyploidy on islands: its emergence and importance for diversification[END_REF], and probably much lower for other Pacific islands, including New Caledonia. The timing of polyploidisation on islands, i.e. pre or post-colonisation, is often difficult to determine [START_REF] Crawford | Genetic diversity in Asteraceae endemic to ocean islands: Baker's law and polyploidy[END_REF][START_REF] Meudt | Polyploidy on islands: its emergence and importance for diversification[END_REF] and is a major problem [START_REF] Johnson | Multiple colonizations, hybridization and uneven diversification in Cyrtandra (Gesneriaceae) lineages on Hawai'i Island[END_REF].

Dispersal, hybridization and polyploidisation are potentially intimately interconnected and may be difficult to tease apart. For example, in grasses, polyploids may be more successful at long-distance dispersal than diploids [START_REF] Linder | Does polyploidy facilitate long-distance dispersal?[END_REF]. Multiple allopolyploid origins likely increased the ecological amplitude of Aegilops triuncialis (Poaceae) during its invasion of California [START_REF] Meimber | Mutliple origins promote the ecological amplitude of allopolyploid Aegilops (Poaceae)[END_REF]. Polyploidy is more likely to follow hybridization when the two parents are distantly related [START_REF] Paun | Hybrid speciation in angiosperms: parental divergence drives ploidy[END_REF]). The distinction between auto-and allotetraploid is not always straightforward [START_REF] Ramsey | Pathways, mechanisms, and rates of polyploid formation in flowering plants[END_REF], but allopolyploids are expected to be more prevalent in nature than autopolyploids [START_REF] Soltis | The role of genetic and genomic attributes in the success of polyloids[END_REF]. There are many examples of endemic species or radiations with a hybrid origin in island plants (Table 6 in [START_REF] Johnson | Multiple colonizations, hybridization and uneven diversification in Cyrtandra (Gesneriaceae) lineages on Hawai'i Island[END_REF], where it is not clear which came first: colonisation or hybridization. In the case of Nicotiana sect. Suaveolentes (Solanaceae), all of which are allotetraploid [START_REF] Chase | Molecular systematics, GISH and the origin of hybrid taxa in Nicotiana (Solanaceae)[END_REF], hybridisation in South America preceeded colonisation, with allotetraploid species reaching French Polynesia, New Caledonia, Namibia (Africa) and Australia about six million years ago [START_REF] Cauz-Santos | Genomic insights into recent species divergence in Nicotiana benthamiana and natural variation in Rdr1 gene controlling viral susceptibility[END_REF]). However, only in Australia has there been a recent radiation into more than 60 species [START_REF] Chase | Species delimitation in Nicotiana sect. suaveloentes (Solanaceae) : reciprocal illumination leads to recognition of many new species[END_REF]. No diploids occur in the range of N. sect.

Suaveolentes, and none of the species of this section occurs within the range of the parental diploids in South America [START_REF] Chase | Molecular systematics, GISH and the origin of hybrid taxa in Nicotiana (Solanaceae)[END_REF]. The dispersal vector is assumed to be birds [START_REF] Mummenhoff | Gone with the bird: Late tertiary and quaternary intercontinental long-distance dispersal and allopolyploidization in plants[END_REF], but the modern wide disjunction between diploid parents and tetraploid offspring is perplexing.

Here, we investigate the origins of New Caledonian Grevillea (Proteaceae), which comprises ten endemic species [START_REF] Majourau | A review of Grevillea (Proteaceae) from New Caledonia with the description of two new species[END_REF] among ca. 360 species in the genus. They are often dominant in New Caledonian maquis on ultramafic rocks [START_REF] Jaffré | Plant communities of the maquis on ultramafic rocks of New Caledonia[END_REF] and are of particular interest because of their tendency to accumulate manganese, which confers to their metal-rich biomass some promising properties for organic chemistry [START_REF] Losfeld | Leaf-age and soil-plant relationships: key factors for reporting trace-elements hyperaccumulation by plants and design applications[END_REF]. The only chromosome count available for this group is that of G. meisneri (n=22, [START_REF] Carr | Chromosome numbers of New Caledonian plants[END_REF], a much higher count than those typically observed in the family (from n = 10 to n = 14, [START_REF] Stace | Did 'Paleo-polyploidy' really occur in Proteaceae?[END_REF]) and for Grevillea in particular (n = 10). With novel molecular phylogenetic data and new genome-size measurements, we sought to determine the number of times Grevillea colonised New Caledonia and obtain new lines of evidence for the origins of polyploidy in this group. Their seeds are also characterised and compared in light of their ecology.

Material and Methods

Sampling, DNA extraction, amplification, cloning and sequencing

Fresh material was collected for each species of New Caledonian Grevillea (G. deplanchei, G. exul, G. gillivrayi, G. macmillanii, G. meisneri (both varieties), G. mondorensis, G. nepwiensis, G. rubiginosa, G. sinuata and G. vuniana). Two accessions of New Caledonian species of the related genus Stenocarpus were also included. All samples were dried in silica-gel [START_REF] Chase | Silica gel: an ideal material for field preservation of leaf samples for DNA studies[END_REF]. Total genomic DNA was extracted with a modified CTAB protocol [START_REF] Doyle | A rapid DNA isolation procedure for small quantities of fresh leaf tissue[END_REF]. Five DNA regions were amplified following the protocols of [START_REF] Mast | A smaller Macadamia from a more vagile tribe: inference of phylogenetic relationships, divergence times, and diaspore evolution in Macadamia and relatives (tribe Macadamieae; Proteaceae)[END_REF]:

(i) plastid atpB, matK, ndhF and the rpl16 intron, and (ii) nuclear PHYA gene.

For each New Caledonian accession, directly sequenced regions of PHYA included multiple single base pair polymorphisms that indicated the presence of two distinct alleles differing at a number of bases without insertions/deletions. These bases were coded following the IUPAC nucleotide ambiguity scheme [START_REF] Cornish-Bowden | Nomenclature for incompletely specified bases in nucleic acid sequences: recommendations 1984[END_REF]. Two combinations of alleles were readily distinguishable, and we separated the alleles by cloning for at least one representative (G. gillivrayi and G. exul) of each combination with pGEM®-T Easy vector (Promega Corp.) according to the manufacturer's instructions. These alleles separated by molecular cloning (five and six clones sequenced) served as guides to reconstruct the two distinct alleles of the other individuals. We kept the IUPAC coding for polymorphisms not found in either cloned accession.

Molecular datasets

The newly generated sequences from the 12 New Caledonian Grevillea (and two Stenocarpus) specimens were combined with molecular data from the study of Mast et al. (2015). In their study, Mast et al. (2015) assessed the phylogenetic relationships of the subtribe Hakeinae (Proteaceae) with a 171 accession molecular dataset (plastid genes atpB, matK and ndhF and the rpl16 intron, and nuclear PHYA) including 97 specimens (representing 93 species) of Grevillea and 55 specimens (one per species) of Hakea, and representatives of other genera of Embothrieae and more distantly related Proteaceae. As more distant outgroups, we followed Mast et al. (2015) and included representatives of Platanaceae (Platanus orientalis) and Nelumbonaceae (Nelumbo lutea). Here, two distinct datasets were assembled: (i) a plastid dataset of atpB, matK, ndhF and the rpl16 intron, and (ii) a nuclear dataset of PHYA sequences. We did not combine the plastid and nuclear datasets because of the presence of distinct alleles in the PHYA sequences of New Caledonian Grevillea. The rpl16 intron sequences varied slightly in length, and we used MAFFT v7 [START_REF] Katoh | MAFFT Multiple sequence alignment software version 7: improvements in performance and usability[END_REF] with default option settings to align them. For all exons (atpB, matK, ndhF and PHYA), we used Mesquite v3.70 [START_REF] Maddison | Mesquite: a modular system for evolutionary analysis[END_REF] to check the reading frame for stop codons. The four combined plastid regions for 185 specimens resulted in a matrix of 6,508 aligned characters (2,191 variable characters of which 1,326 are potentially parsimony informative), whereas for PHYA, the dataset consisted of 174 sequences (including two sequences for each of 11 New Caledonian accessions) and 1,169 aligned characters (593 variable characters of which 374 are potentially parsimony informative). GenBank accession numbers and voucher information for all corresponding sequences are presented in Table S1.

Phylogenetic analyses

For both datasets, maximum likelihood (ML) analyses included a priori division in partitions.

The plastid dataset was divided into 10 partitions, with three partitions (one per codon position) for each gene (atpB, matK and ndhF) and one for the rpl16 intron. The nuclear dataset was divided into three partitions, one for each codon position. Following Mast et al. (2015), trees were rooted with Nelumbo lutea. Maximum likelihood analyses were conducted with IQ-TREE v2 [START_REF] Minh | IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era[END_REF]. For each dataset, the best-score ML tree was inferred with a heuristic search with 500 random-addition replicates and the following settings: random-starting tree, thorough hill-climbing nearest neighbour interchange (NNI) search (-allnni option), perturbation strength of 0.2 (-pers 0.2 option), partition-resampling strategy (--sampling GENE option), and best partition scheme allowing the merging of partitions (-m MFP+MERGE option). Support of nodes was assessed with 1000 replicates for both SH-like approximate likelihood ratio tests (SH-aLRT, [START_REF] Guindon | New algorithms and methods to estimate Maximum-Likelihood phylogenies: assessing the performance of PhyML 3.0[END_REF]) and ultrafast bootstrap (uBV, [START_REF] Minh | Ultrafast approximation for phylogenetic bootstrap[END_REF]. Nodes supported by SH-aLRT ≥ 80 and uBV ≥ 95 were considered strongly supported following authors' recommendations.

Dating analyses

Dating analyses were conducted on the plastid dataset using Bayesian relaxed clocks as implemented in BEAST v1.10.4 [START_REF] Suchard | Bayesian phylogenetic and phylodynamic data integration using BEAST 1.10[END_REF]. Here, we used the primary fossil calibrations of Mast et al. (2015) including the same normal distributions to constrain the ages of the most recent common ancestors (MRCA) of: (i) Proteaceae and Platanaceae (mean of 112.3 and a standard deviation of 6.48), and (ii) Embothrium and Telopea (mean of 37.5 and a standard deviation of 1.07). To properly implement the corresponding rates of evolution, we relied on a calibration strategy of four uncorrelated lognormal clocks, one for each plastid marker. We further used as a guide tree the results of ML plasid analyses. The tree model was set to a birth-death speciation process, which is adequate to analyze datasets with a mixture of inter-and intraspecies sampling [START_REF] Ritchie | The impact of the tree prior on molecular dating of data sets containing a mixture of inter-and intraspecies sampling[END_REF]. The BEAST analysis consisted of 50 million generations of MCMC with the parameters and trees sampled every 5,000 generations. A burn-in of 25% was applied after checking the loglikelihood curves. The maximum credibility tree, median ages and their 95% highest posterior density (HPD) were generated with TreeAnnotator v1.10.4, which is part of the BEAST software package. Convergence of runs was then assessed graphically by examining the ESS of relevant parameters under Tracer v.1.7 [START_REF] Rambaut | Posterior summarization in bayesian phylogenetics using Tracer 1.7[END_REF].

Genome-size estimation

Fresh leaves from five individuals of Grevillea gillivrayi and G. rubiginosa were collected in the field and G. robusta from cultivated trees in Noumea. Nuclei were prepared with the following isolation buffer: 45 mM MgCl 2 , 30 mM sodium citrate, 60 mM MOPS pH 7.0, 1% PVP 10.000, 0.1% Triton X-100 and 10 mM sodium metabisulfite (S 2 O 5 Na 2 ) [START_REF] Bourge | Flow cytometry as tool in plant sciences, with emphasis on genome size and ploidy level assessment[END_REF]) and stained with propidium iodide 50 μg/mL. The samples were run on a CytoFLEX S Cytometer (Beckman Coulter), excitation 561nm, 46 mW; emission through a 690/50 nm band-pass filter. The following standards were used: Solanum lycopersicum "Roma" (2C = 1.99 pg) and Petunia hybrida PxPc6 (2C = 2.85 pg) [START_REF] Marie | Paraphyly changes understanding of timing and tempo of diversification in subtribe Hakeinae (Proteaceae), a giant Australian plant radiation[END_REF].

Seed morphology

Mature fruits of Grevillea exul, G. rubiginosa and G. gillivrayi were harvested from plants growing in the south of New Caledonia. Seeds were extracted after fruit drying. For each species, 20 seeds were weighed and measured (entire seed and embryo length and width), and average dimensions were determined. Embryo length:seed length (El:Sl) ratio and embryo width:seed width (Ew:Sw) ratio were calculated.

Results

Phylogenetic analyses

Overall, the plastid ML tree is well supported (Figure 1) with 120 of the 184 nodes supported by both SH-aLRT ≥ 80% and uBV ≥ 95%. Grevillea is paraphyletic because representatives of Hakea are sister (SH-aLRT 78.7%, uBV 92%; always given in this order below) to 45 Grevillea species. New Caledonian Grevillea species form two well-supported (≥ 80%, ≥ 95%) clades within group 3 sensu Mast et al. (2015): the exul group (G. exul, G. macmillanii, G. rubiginosa and G. sinuata) and the gillivrayi group (G. deplanchei, G. gillivrayi, G. meisneri, G. mondorensis, G. nepwiensis and G. vuniana). Within the gillivrayi group, our sample of G.

gillivrayi fails to pair with the sample of G. gillivrayi in Mast et al. (2015). The exul group is sister ( 98.2%, 100%) to a pair of south-western Australian species, G. infudibularis and G. tripartita (the oncogyne group sensu [START_REF] Makinson | Proteaceae 2. Grevillea. Flora of Australia[END_REF]. Sister to the gillivrayi group is the Sulawesi endemic G. elbertii (98.8%, uBV of 100%) followed by Melanesian G. chloroxantha (alternatively placed in the genus Finschia) (73.2%, 98%).

In the PHYA ML tree (Figure 2), overall support is lower, and only 63 nodes of the 173 nodes are supported by both SH-aLRT ≥ 80% and uBV ≥ 95%. The inferred topology is similar to the plastid tree, with some noticeable differences, i.e., the Hakea species form two wellsupported clades instead of one (100%, 100%). The direct sequencing of PHYA produced many loci with two bases (polymorphisms) and thus indicates that all New Caledonian Grevillea have two distinct copies. These alleles form two distinct groups in the PHYA tree (Figure 2). The first allele is weakly supported (0%, 83%) as sister (100%, 100%) to most representatives of group 3 sensu Mast et al. (2015), whereas the second is embedded (0%, 99%) among representatives of group 4 sensu Mast et al. (2015). In both plastid and PHYA trees, the two New Caledonian accessions of Stenocarpus (S. milnei and S. rubiginosa) are sister to Australian S. sinuatus and Strangea cynanchicarpa. Thus, Stenocarpus is paraphyletic to Strangea.

Dating analyses

Post-burn-in parameters for the BEAST analysis had ESS values ≥ 200. Based on the plastid dataset, the origin of Grevillea/Hakea occurred during the Eocene with a median age of ca.

38.2 million years ago (Mya; 95% HPD: 32.1-44.9 Mya; Figure 3). The estimated ages of the two New Caledonian clades are largely overlapping. The plastid haplotype of the exul group diverged from those of G. infundibularis + G. tripartita ca. 12.9 Mya (95% HPD: 9.7-16 Mya) with a crown age of ca. 10.2 My (95% HPD: 7.3-13.2 My). The plastid haplotype of the gillivrayi group diverged from those of G. elbertii ca. 13.2 Mya (95% HPD: 7.3-13.2 Mya) with a crown age of ca. 9.2 My (95% HPD: 6.5-12.2 My).

Genome-size estimation

We obtained the following genome size estimates: G. gillivrayi, mean 1C = 1.84 pg (standard Petunia hybrida PxPc6); G. robusta with mean 1C = 0.92 pg (standard Petunia hybrida PxPc6), and G. rubiginosa, mean 1C = 1.60 pg (standard = Solanum lycopersicum "Roma").

Seed morphology

The seeds of Grevillea exul, G. rubiginosa or G. gillivrayi present a common structure of a winged testa with the integument surrounding the central embryo (Figure 4). There is some size variation between species in wing size (the largest in G. exul) and seed color (bright for G. gillivrayi, dark in both G. exul and G. rubiginosa, the last sometimes black). Sections of seeds revealed that they are all constituted by a large embryo that occupies all space in the central portion. The embryo length:seed length (El:Sl) ratio is greather than 40% in all cases.

No endosperm was detected.

In contrast, there are significant differences in seed size, shape and weight between species (Table 1). Grevillea rubiginosa and G. exul seeds are round, the latter smaller even though it is the heaviest of all studied species. Grevillea gillivrayi seeds are more oval than the others with an average weight close to that of G. rubiginosa. Finally, considering the space occupied by the embryo, i.e. El:Sl and Ew:Sw ratios, the largest embryo clearly belongs to G. gillivrayi, whereas G. exul has the smallest.

Discussion

Polyploidy in Proteaceae

Our genome-size estimate for Grevillea robusta is larger than that reported previously (1C = 0.92 pg vs. 0.80 pg, [START_REF] Ohri | Nuclear DNA amounts in some tropical hardwoods[END_REF]. The other Grevillea estimate available is for G. australis, (1C = 1.21 pg, [START_REF] Jordan | Environmental adaptation in stomatal size independent of the effects of genome size[END_REF]. The estimates for the New Caledonian endemics G. gillivrayi (1C = 1.84 pg) and G. rubiginosa (1C = 1.60 pg) are therefore 2 times and 1.74 times larger than that of G. robusta, respectively. [START_REF] Stace | Did 'Paleo-polyploidy' really occur in Proteaceae?[END_REF] reported n = 10 in ten species of Grevillea, whereas New Caledonian G. meisneri is n ≈ 22 [START_REF] Carr | Chromosome numbers of New Caledonian plants[END_REF].

These counts and genome-size estimates indicate that the New Caledonian species of Grevillea are likely to be tetraploids. Their genome sizes are larger than those otherwise observed in Hakeinae (1C, μ = 0.99; σ = 0.19) and Grevilleoideae (1C, μ = 1.19; σ = 0.41) [START_REF] Pellicer | The Plant DNA C-values database (release 7.1): an updated online repository of plant genome size data for comparative studies[END_REF].

Genome sizes are comparatively small in Proteaceae, except for Persoonioideae (1C, μ = 23.71) [START_REF] Jordan | Environmental adaptation in stomatal size independent of the effects of genome size[END_REF][START_REF] Pellicer | The Plant DNA C-values database (release 7.1): an updated online repository of plant genome size data for comparative studies[END_REF]. [START_REF] Stace | Did 'Paleo-polyploidy' really occur in Proteaceae?[END_REF] reported only four cases of polyploidy in Proteaceae: Franklandia triaristata (Western Australia), Dilobeia thouarsii (Madagascar), Persoonia (=Toronia) toru (New Zealand) and Grevillea meisneri.

Small genome sizes in Proteaceae would be expected considering their notorious ability to cope with low phosphorus soils and multiple unique traits associated with P-economy, including P-scavenging cluster roots, efficient P-remobilization from senescing organs, substitutes to membrane phospholipids etc. [START_REF] Lambers | Phosphorus nutrition in Proteaceae and beyond[END_REF]. The distinctiveness of the larger genomes reported for Persoonioideae [START_REF] Jordan | Environmental adaptation in stomatal size independent of the effects of genome size[END_REF] and lack of proteoid roots [START_REF] Lamont | Mechanisms for enhancing nutrient uptake in plants, with particular reference to mediterranean South Africa and Western Australia[END_REF]) are noteworthy. Polyploidy in the New Caledonian species of Grevillea, which are restricted to ultramafic substrates, thus appears counter-intuitive. These soils are particularly P-impoverished, and a previous study suggested than these substrates favoured smaller genomes in the western Balkans [START_REF] Pustahija | Small genomes dominate in plants growing on serpentine soils in West Balkans, an exhaustive study of 8 habitats covering 308 taxa[END_REF]. Genome sizes are small in all Grevillea species, regardless of their ploidy.

Biogeography and timing of insular allopolyploids

Our phylogenetic results indicate a hybrid origin of New Caledonian Grevillea involving two or more, probably three, parental species. All New Caledonian Grevillea have a plastid genome similar to that of the Grevillea species in group 3 (sensu Mast et al. 2015). However, they form two clades: the exul group sister to a pair of south-western Australian species (G. infudibularis, G. tripartita, the oncogyne group sensu [START_REF] Makinson | Proteaceae 2. Grevillea. Flora of Australia[END_REF] and the gillivrayi group sister to G. elbertii, described from Sulawesi (Sleumer 1955a). This last species is known from only a few collections and localities, including Kabaena and Malili, where it grows in "dry-bush formation on crystalline slate" (Sleumer 1955b), which is likely to be ultramafic.

All New Caledonian Grevillea have two copies of single-copy nuclear PHYA. Mast et al. (2015) included a single accession of a New Caledonian species of Grevillea, G. gillivrayi, and produced a single sequence, which could be explained by the fact that they cloned all their PCR products prior to sequencing. Thus, they likely sequenced just a single clone per accession (and missed the evidence of more than one allele per accession), whereas we directly sequenced our amplification products. They also did not produce a PHYA sequence for G. elbertii. In general, the PHYA tree was weakly supported, but the alleles were separated in two groups, one sister to most representatives of group 3 and another embedded within group 4 (sensu Mast et al. 2015) with the G. huegelii and G. heliosperma clades and G. robusta (all Australian).

The phylogenetic results together with the cytogenetic data indicate an allotetraploid origin for the species of New Caledonian Grevillea, involving Australian diploid species in clades 3 and 4 (sensu Mast et al. 2015). Although it has apparently not been investigated specifically in Proteaceae, plastid genomes in Grevillea and related genera are likely to be maternally inherited, as in most angiosperms [START_REF] Corriveau | Rapid screening method to detect potential biparental inheritance of plastid DNA and results for over 200 angiosperm species[END_REF]. Since the New Caledonian species have the plastid genome of clade 3 (sensu Mast et al. 2015), a species of clade 3 likely was the maternal parent. The fact that G. exul and G. gillivrayi groups occur in two separate clades in the plastid tree suggests that two different maternal parents were involved, but this seems to be contradicted by the PHYA tree in which these two alleles fell in a single clade. However, in PHYA variation is lower, and the accession of G. elbertii is missing, so this result could be an artefact due to these two factors.

Phylogenetic and cytogenetic results suggest one or two allotetraploid origins of the New Caledonian Grevillea species. Their parents were one or two lineages of clade 3 (from Australia and possibly Sulawesi) and one from clade 4 (Australia). New Caledonian Grevillea can therefore be added to the list of island endemics with an hybrid origin involving nonnative parents [START_REF] Johnson | Multiple colonizations, hybridization and uneven diversification in Cyrtandra (Gesneriaceae) lineages on Hawai'i Island[END_REF]. Two scenarios are possible. One implies two or three dispersal events from Australia (or Sulawesi) to New Caledonia, followed by hybridisation/polyploidization and local extinction of the parental diploids, whereas the other scenario would include hybridization/polyploidization in Australia and their dispersal to New Caledonia followed by extinction of the tetraploid progenitors in Australia.

The prime ecosystem for both New Caledonian Grevillea clades is maquis on ultramafic substrates. Their crown ages (c. 10.2 and 9.2 My) is older than that of New Caledonian Dracophyllum (5.2 Ma, [START_REF] Wagstaff | Origin, diversification, and classification of the Australasian genus Dracophyllum (Richeeae, Ericaceae)[END_REF], another group mostly confined to this open ecosystem. Considering their important botanical diversity [START_REF] Isnard | How did the ultramafic soils shape the flora of the New Caledonian hotspot?[END_REF], maquis probably have had a long history before human arrival on the island.

Contrast between the two New Caledonian lineages

The two clades of New Caledonian Grevillea species are readily separable morphologically.

The four species of the exul group all have unilateral inflorescences, white flowers and more or less spherical fruits, whereas the six species of the gillivrayi group have cylindrical (to spherical) inflorescences, non-white flowers (generally reddish, but sometimes pale pink, orange or yellow), and laterally flattened fruits. Our study on seed shapes also revealed a common trait between G. exul and G. rubiginosa, both rounded, in contrasts with the oval form of G. gillivrayi, which is like that of G. meisneri (1.8 × 0.3 mm) according to [START_REF] Jaffré | Plantes de Nouvelle-Calédonie permettant de révégétaliser des sites miniers[END_REF]. Most species are maquis shrubs or less commonly small trees in low forest on ultramafic rocks (peridotites and serpentinites). The species of the exul group have a stronger pioneer behavior, being the first ligneous species capable of growing on ancient nickel mines [START_REF] Jaffré | Composition chimique élémentaire des tissus foliaires des espèces végétales colonisatrices des anciennes mines de nickel en Nouvelle-Calédonie[END_REF]. They all have simple entire leaves, similar to those of G. elbertii (Sleumer 1955b), unlike the pinnatifid leaves of G. robusta.

All New Caledonian Grevillea species are manganese accumulators [START_REF] Jaffré | Accumulation du manganèse par les Protéacées de Nouvelle-Calédonie[END_REF], with some populations of G. meisneri from extreme northern New Caledonia (Massif of Tiébaghi) qualifying as manganese hyperaccumulators (Losfeld et al. 2015, Pillon et al. unpublished). This means that these plants (hyper-)accumulate high quantities of manganese in their leaves (ranging from several thousands to more than 10,000 µg g -1 ). In both G. meisneri and G. exul s.l., manganese was found to be most highly concentrated in their epidermal cells [START_REF] Fernando | Novel pattern of foliar metal distribution in a manganese hyperaccumulator[END_REF][START_REF] Bihanic | Manganese distribution in the Mn-hyperaccumulator Grevillea meisneri from New Caledonia[END_REF], mostly on the lower epidermis in G. meisneri, whereas it is in the upper epidermis in G. exul. Contrary to what has been found in several Mn accumulators or hyperaccumulators, including species in some other genera of Proteaceae [START_REF] Fernando | Manganese accumulation in the leaf mesophyll of four tree species: a PIXE/EDAX localization study[END_REF], Mn was not primarily concentrated in the palisade mesophyll in the Grevillea species studied so far. In G. meisneri, Mn was also found in other tissues, including spongy mesophyll and vascular strands, but in much lower concentration.

Overall, localisation of Mn in G. exul and G. meisneri, representing the two groups of New Caledonian Grevillea, appears similar.

Variation with each group

The exul and gillivrayi groups also differ in their taxonomic, morphological and ecological diversity. The four species of the exul group are relatively similar to one another, differing in inflorescence and tepal pubescence (from glabrous in G. sinuata to pale pubescent in G. exul and G. macmillanii and red in G. rubiginosa) and inflorescence structure (mostly simple racemes except in G. rubiginosa in which they may be ramified and composed of up to five racemes). The leaves of G. macmillanii are long, narrow and almost needle-like, whereas those of G. rubiginosa are relatively broad and often covered with reddish hairs underneath.

All have a similar ecology in relatively disturbed maquis on ultramafic substrates, with only G. rubiginosa also at high elevation, up to 1600 m. The four species have largely nonoverlapping distributions, and allopatry has likely been the main driver of divergence.

The six species of the G. gillivrayi group display greater diversity. Grevillea meisneri and G. vuniana have pendent almost spherical inflorescences. Grevillea meisneri is a specialist of relatively dry maquis and low forest mostly on serpentinites in the north-west of the main island, whereas G. vuniana is a rare species known only from low, wet forest at a single location (Yaté) in the south-east. Other species have cylindrical and ± horizontal inflorescences and are found in disturbed to pristine maquis on peridotites. Grevillea gillivrayi has a peculiar architecture, with a drastic decrease in branching vigour and intensity along the trunk and inflorescences born on spindly erect axes [START_REF] Salmon | L'architecture végétale au service de l'écologie fonctionnnelle : investigation au sein du genre Grevillea (Proteaceae) de Nouvelle-Calédonie[END_REF], similar to G. deplanchei. The two species have broadly overlaping distributions in the southern half of Grande Terre and the Isle of Pines, but G. deplanchei is a riparian species with typical stenophyllous leaves. Grevillea nepwiensis is isolated in the north-western region with inflorescences covered by red pubescence, whereas G. mondorensis is known from a single location (Mont Dore) and has glabrous inflorescences borne on older wood, sometimes on trunks (cauliflory). Therefore, in addition to allopatry, factors such as pollination syndromes (inflorescence orientation and position) and ecology (river, substrate), probably triggered diversification of this group. It should also be expected that interspecific hybridization/allopolyploidization and environmental stress (e.g., ultramafic substrates) would activate (retro)transposons [START_REF] Grandbastien | Stress activation and genomic impact of Tnt1 retrotransposons in Solanaceae[END_REF][START_REF] Parisod | Impact of transposable elements on the organization and function of allopolyploid genomes[END_REF], which could also be generating interspecific reproductive barriers in these species of Grevillea through genomic structural changes making hybrids less fertile or inviable.

Seed morphology

Seed morphology of G. exul, G. rubiginosa and G. gillivrayi revealed common traits such as a wing, recognized for several New Caledonian Geissois species (Cunoniaceae) as essential for wind dispersal. [START_REF] Olde | The Grevillea Book[END_REF] classified these species as exhibiting the winged seed type, in which a membranous outgrowth of the testa forms a wing that completely surrounds the seed, and the two others are of the oat-style seed, which lacks the membranous wing but instead has revolute margins and ellipsoidal/hemispherical seeds with a convex outer surface and a flat inner surface. Dispersal by anemochory can provide a real advantage in an open, nutrient-poor environment [START_REF] Fenner | The ecology of seeds[END_REF]. Indeed, in such an environment, the distance of the seeds from the mother plant limits competition for mineral resources. From this point of view, G. exul with its larger wing has an advantage, which is reinforced by the non-dormant seed type of our studied species (sensu [START_REF] Baskin | A classification system for seed dormancy[END_REF]) Seeds of these species all germinate in less than 30 days, the first appearing roughly 10 days in favorable conditions (L 'Huillier et al. 2010). This characteristic also occurs in the southwestern Australian G. synaphae [START_REF] Sweedman | Australian seeds: a guide to their collection, identification and biology[END_REF], a member of group 3 (Mast et al. 2015), and could be associated with winged seed type [START_REF] Olde | The Grevillea Book[END_REF], indicating affinities with these three New Caledonian species, and also with G. meisneri (personal observation). In contrast, the seeds of G. sericea and G. buxifolia (both group 4, Mast et al. 2015), defined as oat-style seed [START_REF] Olde | The Grevillea Book[END_REF], both have physical dormant seed [START_REF] Briggs | Investigations into seed dormancy in Grevillea linearifolia, G. buxifolia and G. sericea: anatomy and histochemistry of the seed coat[END_REF]. The fact that New Caledonian species germinate easily is due in a large part to the fact that they have exalbuminate seeds with a large embryo. Several authors have studied the embryo morphology [START_REF] Forbis | The evolution of embryo size in angiosperms and other seed plants: implicattions for the evolution of seed dormancy[END_REF][START_REF] Baskin | Seeds: ecology, biogeography, and evolution of dormancy and germination[END_REF], and all agree that underdeveloped embryos are primitive and mature embryos advanced because germination does not alone guarantee plantlet establishment. To optimize this, embryo size and reserve type are critical factors. Grevillea exul and G. rubiginosa are known to possess oleaginous seeds [START_REF] Bombarda | Fatty acids profile of Alphitonia neocaledonica and Grevillea exul var. rubiginosa seed oils, occurrence of an omega 5 series[END_REF], and lipids produce twice as much energy as carbohydrates for the same mass, which confers to both these species the ability to colonize open areas quickly and efficiently. Moreover, G. exul seeds, with an average weight 1.6 times that of G. rubiginosa despite being almost the same size, seems to be even better adapted.

Conclusions

The ten species of New Caledonian Grevillea may descend from one or two hybridization events involving two or three parental diploid taxa that colonized the island independently but are now extinct. They may represent the remnants of a hybrid swarm that formed 9-13 Mya, when these diploids came into contact in a new, challenging environment. It is possible that allotetraploidy helped these plants adapt and diversify on the ultramafic substrates of New Caledonia via the lineage-specific ohnologue resolution (LORe) model [START_REF] Robertson | Lineage-specific rediploidization is a mechanism to explain time-lags between genome duplication and evolutionary diversification[END_REF], in which the redundant, modular structure of duplicated gene regulatory networks offers polyploid species increased possibilities for evolutionary innovation and adaptation via novel substitutions in duplicated genes. Multiple colonizations and hybridisations may therefore be under-reported processes that could play an important role in the genesis of island radiations. Reticulate evolution will have to be taken into account when considering taxonomic concepts in subtribe Hakeineae.

(

  G. macmillanii, G. rubiginosa and G. sinuata, from the top left and then down) and the gillivrayi group (G. deplanchei and G. gillivrayi, from top to bottom, in that order), respectively, are shown for illustration (all photographs by Y. Pillon).

Figure 2 .

 2 Figure 2. PHYA tree. Maximum likelihood tree from analysis of the nuclear gene PHYA. To increase the prominence of the New Caledonian taxa, only a fraction of the outgroup taxa is figured and Hakea species are lumped. Clade support is indicated by circles on nodes. New Caledonian Grevillea species are highlighted in blue (exul group) or red (gillivrayi group). On the left, photographs of whole specimens of species belonging to the exul group (G. macmillanii, G. rubiginosa and G. sinuata, from the top left and then down) and the gillivrayi group (G. deplanchei and G. gillivrayi, from top to bottom, in that order), respectively, are shown (all photographs by Y. Pillon).

Figure 3 .

 3 Figure 3. Dated tree. Dated phylogenetic tree resulting from the BEAST analyses of the plastid dataset. To increase the prominence of the New Caledonian taxa, outgroup taxa are not figured and Hakea species are lumped. Horizontal bars on nodes represent 95% HPD of age estimates. New Caledonian Grevillea species are highlighted in blue (exul group) or red (gillivrayi group), with arrow pointing to the crown nodes. Time-scale (horizontal) in Mya (Plio.: Pliocene, Plei.: Pleistocene). At the bottom, photographs displaying fruits from species belonging to the exul group (G. rubiginosa, first picture on the left) and the gillivrayi group (G. deplanchei and G. nepwiensis, from left to right), respectively, are shown (all photographs by Y. Pillon).

Figure 4 .

 4 Figure 4. Seeds of Grevillea exul, G. rubiginosa and G. gillivrayi.

Table 1 .

 1 Seed characteristics of Grevillea exul, G. rubiginosa and G. gillivrayi (n=20) Plastid tree. Maximum likelihood tree from analysis of the plastid dataset. To increase the prominence of the New Caledonian taxa, only a fraction of the outgroup taxa is figured and all Hakea species are lumped. Clade support is indicated by circles on nodes.New Caledonian Grevillea species are highlighted in blue (exul group) or red (gillivrayi group). On the left, photographs of inflorescences from species belonging to the exul group

		G. exul	G. rubiginosa	G. gillivrayi
	Seed average weight (mg ± s.d.)	24.5 ± 1.7	15.7 ± 1.2	16.9 ± 1.9
	Seed average length (mm ± s.d.)	12.9 ± 1.4	11.6 ± 1.1	15.6 ± 1.8
	Embryo average length (mm ± s.d.)	5.2 ± 0.6	5.9 ± 0.7	9.4 ± 0.9
	Embryo length:seed length (El:Sl) ratio	40.3%	50.9%	60.2%
	Seed average width (mm ± s.d.)	9.6 ± 1.3	8.3 ± 0.8	7.0 ± 1.0
	Embryo average width (mm ± s.d.)	3.9 ± 0.5	3.6 ± 0.6	4.1 ± 0.5
	Embryo width:seed width (Ew:Sw) ratio	40.6%	43.4%	58.6%

Table S1 .

 S1 List of DNA sequences produced for this study with voucher information and GenBank accession numbers.

					GenBank accession number
	species	specimen_voucher	atpB	mat K	ndhF	PHYA	rpl16
	Grevillea cf gillivrayi YP1475	Pillon & Bruy 1475	OP589202 OP589215 OP619967 OP619975&OP619976 OP675548
	Grevillea deplanchei	Pilllon, Desnues & Isnard 1471 OP589203 OP589216 OP619963 OP619977&OP619978 OP675549
	Grevillea exul	Pillon & Nigote 1477	OP589204 OP589217 OP619968 OP619979&OP619980 OP675550
	Grevillea gillivrayi YP1472	Pillon 1472	OP589205 OP589218 OP619964 OP619981&OP619982 OP675551
	Grevillea macmillanii	Pillon 1486	OP589206 OP589219 OP619969 OP619983&OP619984 OP675552
	Grevillea meisneri var meisneri	Pillon 1469	OP589207 OP589220 OP619970	-	OP675553
	Grevillea meisneri var rhododesmia Pillon & Poullain 1463	OP589208 OP589221 OP619971 OP619985&OP619986 OP675554
	Grevillea mondorensis	Pillon 1460	OP589209	-	OP619965 OP619987&OP619988 OP675555
	Grevillea nepwiensis	Pillon, Poullain & Fleurot 1464 OP589210 OP589222 OP619966 OP619989&OP619990 OP675556
	Grevillea rubiginosa	Pillon & Isnard 1481	OP589211 OP589223 OP619962 OP619991&OP619992 OP675557
	Grevillea sinuata	Pillon & Bruy 1474	-	-	-	OP619993&OP619994 OP675558
	Grevillea vuniana	Pillon & al 1511	OP589212 OP589224 OP619972 OP619995&OP619996 OP675559
	Stenocarpus milnei	Pillon 1488	OP589213 OP589225 OP619973	OP619997	OP675560
	Stenocarpus rubiginosus	Pillon 1491	OP589214 OP589226 OP619974	OP619998	OP675561

Acknowledgements

We thank David Bruy, Dominique Fleurot, Malik Oedin, Cyril Poullain, Jean-Pierre Butin and the RLA collective for assistance in the field. We thank the Southern and Northern Provinces of New Caledonia for issuing collection permits (APA_NCPS_2018_014). Mickaël Bourge and Nicolas Valentin (I2BC) made the cytological studies, and Sylvain Merlot provided logistical support. We thank two anonymous reviewers for insightful comments on an earlier version of the manuscript.

Funding

This work (ID 1702-017) was publicly funded through ANR (the French National Research Agency) under the "Investissements d'avenir" program with the reference ANR-10-LABX-001-01 Labex Agro and coordinated by Agropolis Fondation under the frame of I-SITE MUSE (ANR-16-IDEX-0006), and the Fondation pour la Recherche pour la Biodiversité.

Data availability statement

All DNA sequences are available in GenBank. Voucher information, accession numbers, complete trees and and scripts are available in supplementary material online.

Disclosure statement

No potential conflict of interest was reported by the authors.

Author contributions

YP, SI, BF, MWC, GJK wrote the paper. PM and KG produced the DNA sequences, GJK conducted the phylogenetic analysis and dating.

Supplementary material.

Figure S1. Plastid tree. Maximum Likelihood tree (with all analyzed taxa) resulting from the analysis of the plastid dataset. To increase the visibility of the tree, a cladogram representation is used. Clade support is provided on nodes with SH-aLRT values on the left and uBV values on the right. New Caledonian Grevillea species are also highlighted using blue branches. 1 . 4 / 8 6 8 6 . 2 / 9 9 0 / 6 2 9 8 / 1 0 0 9 8 . 8 / 1 0 0 9 9 . 4 / 1 0 0 9 9 . 9 / 1 0 0 0 / 7 9 9 8 . 6 / 1 0 0 0 / 9 1 9 1 / 9 5 0 / 9 5 8 5 . 5 / 9 9 7 5 . 3 / 1 0 0 9 7 . 8 / 1 0 0 9 6 . 2 / 1 0 0 9 9 . 9 / 1 0 0 9 9 . 6 / 1 0 0 7 3 . 9 / 9 7 9 2 / 9 9 9 0 . 6 / 1 0 0 7 7 . 6 / 9 8 9 6 . 3 / 1 0 0 9 8 . 1 / 1 0 0 9 9 . 9 / 1 0 0 1 0 0 / 1 0 0 6 5 . 4 / 9 0 7 1 . 6 / 9 7 9 7 / 1 0 0 7 1 . 9 / 9 9 9 1 . 9 / 9 8 9 9 . 5 / 1 0 0 9 5 . 4 / 1 0 0 9 9 . 2 / 1 0 0 9 7 . 9 / 1 0 0 7 4 . 9 / 8 5 9 7 / 1 0 0 9 1 . 1 / 9 5 9 1 . 1 / 1 0 0 9 6 / 1 0 0 7 5 / 9 7 9 6 / 9 9 9 7 . 9 / 1 0 0 9 5 . 7 / 1 0 0 9 3 . 4 / 1 0 0 9 6 . 4 / 1 0 0 9 0 . 1 / 1 0 0 9 9 . 4 / 1 0 0 9 8 . 6 / 1 0 0 9 9 . 4 / 1 0 0 7 4 . 5 / 9 9 8 9 . 8 / 9 9 9 9 . 1 / 1 0 0 0 / 8 9 0 / 2 8 9 4 / 9 8 9 2 . 9 / 9 6 9 0 . 8 / 6 5 3 2 . 5 / 6 7 1 0 0 / 1 0 0 9 8 . 6 / 1 0 0 1 0 0 / 1 0 0 9 1 / 1 0 0 7 5 / 9 8 9 7 . 6 / 1 0 0 7 0 . 6 / 8 9 9 4 . 4 / 1 0 0 6 0 . 9 / 7 7 9 6 . 5 / 9 9 9 5 . 8 / 1 0 0 7 5 / 1 0 0 0 / 8 0 7 4 . 6 / 1 0 0 9 9 . 4 / 1 0 0 8 9 . 9 / 1 0 0 0 / 9 3 9 0 . 3 / 1 0 0 7 8 / 3 7 7 5 / 1 0 0 9 8 . 9 / 1 0 0 9 9 . 2 / 1 0 0 9 3 . 9 / 1 0 0 9 9 . 2 / 1 0 0 8 8 . 1 / 1 0 0 7 3 . 3 / 9 8 9 9 . 9 / 1 0 0 9 9 . 5 / 1 0 0 9 0 . 5 / 1 0 0 9 5 . 3 / 1 0 0 0 / 7 7 1 0 0 / 1 0 0 0 / 8 1 9 5 . 3 / 1 0 0 9 3 . 9 / 1 0 0 9 1 . 6 / 1 0 0 9 7 . 9 / 1 0 0 9 9 . 9 / 4 5 6 2 . 3 / 9 6 9 9 . 5 / 1 0 0 9 4 . 9 / 1 0 0 7 4 . 9 / 1 0 0 7 8 . 7 / 1 0 0 9 9 . 6 / 1 0 0 9 9 . 5 / 1 0 0 7 4 . 6 / 1 0 0 9 1 . 3 / 1 0 0 9 1 . 4 / 6 3 9 6 . 6 / 1 0 0 0 / 8 6 9 3 . 5 / 9 9 9 4 . 9 / 1 0 0 7 3 . 2 / 9 8 8 8 . 5 / 8 5 0 / 6 6 9 9 . 2 / 1 0 0 9 9 . 3 / 1 0 0 8 9 . 2 / 1 0 0 9 1 / 1 0 0 8 9 . 7 / 9 9 9 3 . 7 / 1 0 0 9 0 . 4 / 1 0 0 9 6 . 2 / 1 0 0 7 1 . 2 / 9 8 1 0 0 / 1 0 0 9 6 . 3 / 1 0 0 7 5 . 9 / 9 9 1 0 0 / 1 0 0 9 7 . 9 / 1 0 0 0 / 8 8 7 4 . 5 / 1 0 0 9 3 / 1 0 0 7 7 / 9 6 9 1 . 1 / 1 0 0 9 8 . 2 / 1 0 0 9 7 . 2 / 1 0 0 0 / 9 3 9 2 . 2 / 1 0 0 9 6 . 5 / 1 0 0 1 0 0 / 1 0 0 7 1 . 7 / 7 5 9 9 . 1 / 1 0 0 9 4 . 8 / 1 0 0 9 8 . 7 / 1 0 0 7 8 . 7 / 6 5 9 7 . 5 / 1 0 0 1 0 0 / 1 0 0 7 4 . 5 / 1 0 0 7 5 / 8 9 7 1 . 7 / 9 9 9 6 / 1 0 0 1 3 . 7 / 8 9 9 0 . 6 / 9 8 7 5 . 4 / 1 0 0 0 / 9 6 4 5 . 2 / 9 9 9 7 . 1 / 1 0 0 8 3 . 4 / 9 9 7 4 . 8 / 9 9 9 0 . 7 / 1 0 0 9 0 . 4 / 9 8 4 6 . 3 / 9 3 0 / 8 9 9 0 . 2 / 9 9 9 7 . 2 / 1 0 0 7 6 . 1 / 1 0 0 9 8 . 4 / 9 8 9 9 . 5 / 1 0 0 0 / 8 7 7 6 . 3 / 1 0 0 9 4 . 6 / 9 2 8 8 . 9 / 9 6 9 0 . 7 / 1 0 0 9 6 . 7 / 1 0 0 5 9 . 4 / 8 7 9 9 . 7 / 1 0 0 7 5 . 2 / 9 0 7 3 . 2 / 9 7 9 4 . 7 / 1 0 0 1 0 0 / 1 0 0 7 8 . 7 / 9 2 0 / 6 2 Figure S2. PHYA tree. Maximum Likelihood tree (with all analyzed taxa) resulting from the analysis of the nuclear gene PHYA. To increase the visibility of the tree, a cladogram representation is used. Clade support is provided on nodes with SH-aLRT values on the left and uBV values on the right. New Caledonian Grevillea species are also highlighted using blue branches. 0 / 8 0 1 0 0 / 1 0 0 7 5 . 9 / 1 0 0 0 / 1 0 0 1 0 0 / 1 0 0 1 0 0 / 1 0 0 0 / 9 8 0 / 5 1 1 0 0 / 1 0 0 0 / 4 9 1 0 0 / 1 0 0 7 5 . 9 / 1 0 0 1 0 0 / 1 0 0 0 / 9 8 0 / 9 8 0 / 6 2 0 / 7 6 1 0 0 / 1 0 0 7 5 . 9 / 9 6 0 / 5 8 5 1 . 2 / 1 0 0 0 / 9 9 0 / 2 2 1 0 0 / 8 6 7 5 . 9 / 1 0 0 7 5 . 9 / 1 0 0 0 / 8 4 7 5 . 9 / 1 0 0 0 / 1 2 1 0 0 / 1 0 0 0 / 2 2 0 / 1 2 7 5 . 9 / 1 0 0 5 1 . 2 / 8 6 0 / 9 5 7 5 . 9 / 1 0 0 1 0 0 / 1 0 0 0 / 6 3 0 / 9 8 7 5 . 3 / 1 0 0 1 0 0 / 1 0 0 7 5 . 9 / 1 0 0 1 0 0 / 1 0 0 1 0 0 / 1 0 0 1 0 0 / 1 0 0 1 0 0 / 1 0 0 0 / 5 6 7 5 . 3 / 1 0 0 1 0 0 / 1 0 0 7 5 . 9 / 1 0 0 5 1 . 2 / 7 1 1 0 0 / 1 0 0 0 / 6 4 1 0 0 / 1 0 0 0 / 8 4 1 0 0 / 1 0 0 1 0 0 / 1 0 0 7 5 . 9 / 1 0 0 0 / 6 0 1 0 0 / 1 0 0 0 / 2 8 1 0 0 / 1 0 0 1 0 0 / 1 0 0 1 0 0 / 1 0 0 1 0 0 / 1 0 0 0 / 6 6 1 0 0 / 8 6 1 0 0 / 1 0 0 0 / 8 0 0 / 5 6 1 0 0 / 1 0 0 0 / 9 9 1 0 0 / 1 0 0 0 / 5 4 1 0 0 / 1 0 0 7 5 . 9 / 1 0 0 7 5 . 9 / 1 0 0 1 0 0 / 1 0 0 1 0 0 / 1 0 0 0 / 5 7 0 / 2 1 0 / 3 8 7 5 . 9 / 9 9 0 / 1 7 7 5 . 3 / 1 0 0 7 5 . 9 / 1 0 0 1 0 0 / 1 0 0 1 0 0 / 1 0 0 1 0 0 / 1 0 0 1 0 0 / 1 0 0 0 / 9 9 0 / 1 6 7 5 . 9 / 1 0 0 0 / 2 4 1 0 0 / 7 5 1 0 0 / 1 0 0 0 / 6 2 0 / 5 7 0 / 9 1 7 5 . 9 / 1 0 0 0 / 5 7 0 / 7 5 5 1 . 2 / 5 2 1 0 0 / 1 0 0 7 5 . 9 / 1 0 0 0 / 1 6 1 0 0 / 1 0 0 1 0 0 / 1 0 0 1 0 0 / 1 0 0 0 / 9 4 1 0 0 / 1 0 0 1 0 0 / 1 0 0 0 / 7 4 1 0 0 / 8 6 7 5 . 9 / 7 5 0 / 9 8 0 / 8 2 7 5 . 9 / 1 0 0 0 / 8 2 1 0 0 / 1 0 0 7 5 . 9 / 1 0 0 1 0 0 / 1 0 0 0 / 8 3 1 0 0 / 1 0 0 7 5 . 3 / 1 0 0 7 5 . 9 / 1 0 0 0 / 2 4 7 5 . 3 / 1 0 0 0 / 4 6 1 0 0 / 1 0 0 7 5 . 9 / 1 0 0 1 0 0 / 1 0 0 1 0 0 / 1 0 0 1 0 0 / 1 0 0 1 0 0 / 1 0 0 0 / 7 7 0 / 3 7 0 / 1 3 0 / 9 9 0 / 9 9 1 0 0 / 1 0 0 1 0 0 / 1 0 0 7 5 . 9 / 1 0 0 1 0 0 / 1 0 0 0 / 4 3 0 / 9 2 7 5 . 9 / 8 6 0 / 7 6 1 0 0 / 1 0 0 7 5 . 9 / 1 0 0 1 0 0 / 1 0 0 1 0 0 / 1 0 0 1 0 0 / 1 0 0 0 / 8 5 0 / 8 0 / 5 3 0 / 5 8 0 / 9 9 0 / 8 3 7 5 . 9 / 1 0 0 1 0 0 / 1 0 0 7 5 . 3 / 7 7 1 0 0 / 1 0 0 0 / 8 1 7 5 . 9 / 1 0 0 7 5 . 9 / 1 0 0 1 0 0 / 1 0 0 7 5 . 3 / 7 7 1 0 0 / 1 0 0 0 / 8 Figure S3. Dated tree. Dated phylogeny (with all analyzed taxa) resulting from the BEAST analyses of the plastid dataset. Median ages are provided on nodes. Horizontal bars on nodes represent 95% HPD of age estimates. New Caledonian Grevillea species are also highlighted using blue branches. Two stars indicate the nodes that are constrained with fossil calibrations.

. 0

Grevillea_australis

-1 7 5 -1 5 0 -1 2 5 -1 0 0 -7 5 -5 0 -2 5 0

Grevillea_rosmarinifolia Grevillea_diversifolia


L Di Pace

F Dacquait

F Nguyen

B Larat

Development of PACTITER code and its application to safety analyses of ITER Primary Cooling Water System

Keywords: ITER, safety analysis, ACPs, PACTITER, PACTOLE

 in the CORELE test facility. The comparison between experimental results and PACTITER predictions has provided good results, revealing the influence of the Reynolds on the material release rate. A new version of PACTOLE (version 3) has been designed to account for recent development relevant to the behaviour of corrosion products: improvement of the corrosion/release model, of the oxide and equilibrium concentration determination. Moreover, the data processing architecture and the numerical analysis for solving transient equations have been totally reviewed. It is foreseen that the PACTITER code will profit by these improvements.

Introduction

The PACTITER (Prediction of ACTivation for ITER) code was originally developed with the name of PACTOLE [START_REF] Beslu | PACTOLE: a calculation code for predicting corrosion products behavior and activation in PWR primary coolant systems[END_REF] at the Commissariat à l'Energie Atomique (CEA) Centre de Cadarache, to predict the formation, activation, migration and deposition of corrosion products in the PWR primary cooling loop. The other main purpose of the code application is estimating the effects of changes in design and operating parameters. The PACTOLE code has been validated by comparison between simulation and more than 20 years of measurements on French PWRs.

2

PACTOLE/PACTITER phenomenological model description According to the models used in version 2 of PACTOLE/PACTITER, the main driving force of base metal release, dissolution and precipitation is the concentration gradient between the surface and the bulk coolant. It is further assumed that the concentration for each element at the pipe wall is very close to its solubility in the same conditions. A deposited element can be dissolved if the element concentration in the bulk water is below the solubility value. When the coolant becomes supersaturated in corrosion products, ions can precipitate on the walls or in the bulk of the fluid to form particles. Equilibrium concentrations are derived from thermodynamic laws and depend on temperature and water chemistry (pH, hydrogen partial pressure). It should be kept in mind that chemical data for PACTOLE are fitted for a reducing environment, i.e. excess concentrations of H 2 are used to prevent radiolysis. This results in very low level of O 2 (< 20 ppb) in the cooling fluid. Precipitates agglomerate by collision to form particles and their size increases up to the limit dependent on degree of turbulence. The agglomerated particles are carried away by the coolant by convection and transported to the wall by mass diffusion. Deposits formed in this way are under the influence of shear stresses from flowing coolant and in some cases release by erosion can occur. Activation occurs when ions and deposits are subjected to neutron flux. On the other hand, the corrosion of structural materials under neutron flux is accompanied by a release of radioactive corrosion products. In addition, the active nuclides (ions and particles) after transportation by the coolant and deposition diffuse inside the oxide layers and contaminate them.

3

Application of PACTOLE to ITER: the development of the PACTITER code The PACTOLE code is currently being used under the name of PACTITER for predicting the Activated Corrosion Product (ACP) inventory in the various ITER (International Thermonuclear Experimental Reactor) heat transfer system (HTS) circuits for accident analysis and personnel safety (ORE assessment). At the beginning (1994)(1995)(1996), the PACTOLE code was adapted as far as the input deck was concerned to the ITER case for predicting activated corrosion products inventory and distribution in the Primary Heat Transfer System (PHTS) of the ITER TCWS (Tokamak Cooling Water System) [START_REF] Di Pace | Activated corrosion products in ITER first wall and shielding blanket heat transfer system[END_REF][START_REF] Nisan | Evaluation of the Activated Corrosion Product Source Term for ITER Heat Transfer System[END_REF]. The major modifications were aimed to simulate the particular ITER PHTS operating conditions, which are quite different from those of PWRs. In PWRs, the coordinated B-Li chemistry is used to control a constant pH 300 °C to a target value of 7.2 characterized by the use of boric acid (H 3 BO 3 ) and Li hydroxide (LiOH) differently from ITER PHTS where only pH and H 2 content are defined. The other main difference is the temperature range, which is 100-240 °C for ITER operation. The other peculiarity of the ITER PHTS is the presence of copper alloys cooled down by pressurized water in the divertor cooling loops. That required adapting the PACTOLE V2 code to consider the new element Cu. Since PACTOLE V2 deals with six chemical elements (Fe, Ni, Mn, Cr, Co and Zr) and ten corresponding isotopes, the introduction of copper into the code required the replacement of Zr by Cu and the addition of related chemical and radiochemical data. The PACTOLE V2 code, incorporating Cu by replacing Zr, was named after as PACTITER V1 and made available in early 1996. It adopted a Cu solubility curve assuming the Cu surface was Cu oxide. This proved to have been an optimistic assumption. These data were obtained by a survey work of the existing literature [START_REF] Eriksson | Aqueous Corrosion of Copper Alloys -Literature Survey[END_REF] and incorporated into the code by CEA Cadarache. The code was then used by ENEA Frascati for its work supporting ITER NSSR-1 (Non-Site Specific Safety Report n.1) in late 1996. The results obtained were considered highly preliminary. In parallel an experimental campaign, performed by CEA Fontenay-aux-Roses, was launched in early 1996 to get Cu solubility data in the ITER divertor PHTS conditions [START_REF] You | Thermodynamic stability and solubility of copper and copper oxides[END_REF]. This experimental study was devoted to determine the valence states of the copper (Cu 0 , Cu + or Cu ++ ) and to obtain solubility in steady state conditions. In order to check the stable phases of Cu in ITER divertor coolant conditions, autoclave tests were performed on copper and copper oxides. These included four tests at 100 and 250 °C on CuO and Cu 2 O, pure copper and Cu-Cr-Zr (the reference Cu alloy for ITER divertor high heat flux components). The major findings of this part of the study were: The related values, illustrated in Figure 1 [START_REF] Di Pace | Development of the PACTITER code and its application to the assessment of the ITER Divertor cooling loop corrosion products[END_REF], are quite different from those adopted first in PACTITER V1 and coming from literature [START_REF] Eriksson | Aqueous Corrosion of Copper Alloys -Literature Survey[END_REF].

Temperature [°C] Cu concentration [µg/kg]

Experimental data

Experimental data correlation Data from literature [START_REF] You | Thermodynamic stability and solubility of copper and copper oxides[END_REF] and literature data [START_REF] Eriksson | Aqueous Corrosion of Copper Alloys -Literature Survey[END_REF] The copper concentrations determined by tests are four or five orders of magnitude larger than the copper solubility obtained by thermodynamic data. Such difference could be explained by:

Figure 1-Cu concentration in steady state conditions, comparison of experiments data

• the validity of thermodynamic data (See Appendix of [START_REF] Beslu | PACTOLE: a calculation code for predicting corrosion products behavior and activation in PWR primary coolant systems[END_REF]);

• the formation of copper colloids during tests;

• the presence of another Cu species, such as copper oxides previously formed during the exposure to vapours at room temperature or before establishing reducing conditions; • the oxidation of Cu due to the existence of oxygen traces in water (copper is known as a very good getter of oxygen). In conclusion, it is possible to state that solubility measured during the tests is questionable.

The new release of the code PACTITER, known as PACTITER V2, incorporating the experimental Cu data was ready in late 1997. It was also updated from the radiochemical viewpoint, to take into account the major radionuclides produced by the copper activation. The three following nuclear reactions involving Cu, found to be the most important ones for safety analysis, were included in PACTITER V2:

-63 Cu(n, γ) 64 Cu; -65 Cu(n, 2n) 64 Cu; -63 Cu(n, α) 60 Co.

Using the new tool PACTITER V2, the ACP assessment for all ITER cooling loops were completed in early 1998 in support to NSSR-2, Volume III "Radiological and Energy Source Terms". The ACP assessment was then repeated to take into account design modification in the PHTS design (e.g. the heat exchangers with primary coolant on the shell side) [START_REF] Di Pace | Final report on Activated Corrosion Products Evaluation for the ITER-FDR PHTS[END_REF]. The comparison with experimental corrosion data related to ITER operating conditions and Cualloys provided a satisfactory result [START_REF] Di Pace | Development of the PACTITER code and its application to the assessment of the ITER Divertor cooling loop corrosion products[END_REF].

Further to the decision taken to update the ITER Final Design Report defined in 1998 (ITER FDR) to reduce size and cost of the machine still satisfying the overall programmatic objective of the ITER EDA Agreement, the machine design was reviewed including that of PHTS during the extension (1998-2001) of the EDA (Engineering Design Activity) phase.

The number of cooling loops of the PHTS was reduced from a total of 20 to 6. Again, the code PACTITER V2 was again applied to estimate the ACP inventory of the ITER PHTS according to the new design in support of accident analysis and worker collective dose assessment, with results documented in the Generic Site Safety Report (GSSR) [8].

ITER PHTS design evolution

Since the first application of the PACTOLE code to assess the ACPs inventory in ITER, the design of the PHTS has evolved along with the other modifications. In Table 1, it is presented the major modifications to ITER PHTS design since 1995. In Figure 2, the ITER PHTS is schematized according to the FDR 1998, together with the PACTITER geometric model of one of the ten loops of the FW/shield/Inboard baffle PHTS.

5

ITER safety requirements for ACPs ACPs will be present in ITER in the various in-vessel and vacuum vessel coolant loops as well as in any coolant loops related to test modules, auxiliary heating or diagnostics equipment. These products impact occupational exposure, routine effluents to the environment, and potential releases during accidents. That has made the ACP inventory evaluation an important task for ITER public and occupational safety. ACPs present in fission reactors cooling loops do not pose a significant public hazard in these installations, while they are a significant hazard to site personnel. In fact, in case of severe accidents in LWRs, ACPs are negligible in terms of radioactive inventory (RI), mobilisable inventory (MI) and environmental source term (EST), compared to the analogous inventories from the reactor core (gas and fission products). On the contrary for fusion devices such as ITER, ACPs could be not negligible in terms of the whole mobilisable inventory (MI) in case of accidents compared to other major source terms, tritium and dust. The enveloping ACPs inventory adopted for ITER accident analyses [START_REF]ITER Safety Analysis Data List, Vers. 4.0.1[END_REF] was assumed to be 10 kg/loop as deposit and 0.6% of deposit mass in the suspended forms (soluble ions or crud particulates), irrespective of the cooling loop type, FW/shield, divertor or vacuum vessel.

The other important safety requirements are the project release guidelines for the ACPs, tritium and dusts as shown in Table 2.

Table 2 -Project Release Guidelines [10]

Events or Conditions

Project Release Guideline(*) Normal Operation event sequences and plant conditions planned and required for ITER normal operation, including some faults, events or conditions which can occur as a result of the ITER experimental nature < 1 g-T as HT and 0.1g-T as HTO and 1 g-metal as AP and 5 g-metal as ACP per year. Incidents deviations from normal operation, event sequences or plant conditions not planned but likely to occur due to failures one or more times during the life of the plant but not including Normal Operation < 1 g-T as HT or 0.1 g-T as HTO or 1 g-metal as AP or 1 g-metal as ACP or equivalent combination of these per event Accidents postulated event sequences or conditions not likely to occur during the life of the plant. < 50 g-T as HT or 5 g-T as HTO or 50 g-metal as AP or 50 g-metal as ACP or equivalent combination of these per event.

Note: (*) HT: elemental tritium (including DT); HTO: tritium oxide (including DTO); AP: divertor or first-wall activation products; ACP: activated corrosion products.

For the occupational radiation exposure (ORE) doses, the following limits and project guidelines have been adopted [10] as shown in Table 3.

Table 3 -ITER limits and project guidelines for ORE doses

Dose Limits ICRP recommended limit for annual individual worker doses 20 mSv averaged over 5 years not to exceed 50 mSv in any year Project Guidelines Annual individual worker doses 5 mSv/year Individual worker dose for any given shift 0.5 mSv/shift

6

Results of ITER ACP assessment by PACTITER As written in section 3, many calculations were carried out with the PACTOLE code first and then with the PACTITER code to assess ACP in the PHTS of ITER TCWS, following the several evolutions of the design. In this section the results presented are referred to the latest evolution of the design as and they are fully documented in the related official technical document [8]. The ACP assessment carried out for a single First Wall/Shield cooling loop provided a value of 1.8 g of suspended cruds and 10.9 g of dissolved ions. The total ACP deposit mass was 1.4 kg/loop. The hazard of the ACP is typically dominated by Mn-56 that arises from Mn and Fe in the base alloy steel, by Co-57, Co-58, that arise from Ni in the base steel and by Co-60 that arises from both Ni and Co in the base steel. The ACP deposit inventory is more than seven times lower than the limit of 10 kg/loop while the ACP inventory in the suspended forms is about 5 times lower than the limit. The ACP assessment for the divertor cooling loop provided values close to the limits, 8.9 kg for the deposit mass and 42 g in the suspended forms. The ACP inventory for VV loops was estimated to be 6 kg/loop for mass deposit and 25.6 g/loop in the suspended forms. It was conservatively assumed that the radiological hazard of VV ACP is 100 times lower compared to the FW/shield ACP. The estimated base metal release rates for the different cooling loops of the ITER TCWS are shown in Table 4. Mass release rates of the VV loop stainless steels are lower due to the lower coolant operating temperature (100 -103 °C) and to the lower coolant velocity < 0.5 m/s in most of the loop. ACP mass and activity inventory were used in accident analyses involving LOCA with consequent releases to the environment. Among several accident sequences analysed [11], a large DV ex-vessel coolant pipe break provided the largest ACP environmental release, equal to 1.2 % of the project release guidelines (see Table 2). The ACP radioactive inventory distribution, assessed by the PACTITER code for ITER TCWS, was used to determine the dose rates around its system components (e.g.; piping, main pumps, valves, heat exchangers) during scheduled and unscheduled maintenance. Starting from the radiation field and taking into account the anticipated work effort, the related annual collective ORE was estimated to be 65 pers-mSv [12] for the first wall/shield loops (30) and divertor loop (35). The contribution from VV loops was not estimated being the related ACP radiation hazard negligible as written before.

PACTITER V3.1: A new version 7.1 Modelling

The PACTOLE V2 code has shown some lacks of modelling [START_REF] Tarabelli | Status and Future Plans of the PACTOLE Code Predicting the Activation and Transport of Corrosion Products in PWR[END_REF]. Therefore, a new code version, namely PACTOLE V3.0, has been designed to account for recent developments relevant to the behaviour of corrosion products in PWR and to help in understanding and modelling the physical-chemical processes [START_REF] Marchetto | PACTOLE V3 : a new code version to predict corrosion product contamination[END_REF]. The PACTOLE V3.0 code is still based on a control volume approach, the primary circuit is represented by an arrangement of several volumes in which transient mass balance equations are solved :

( ) ∑ ∑ - = - + ∂ ∂ Source Sink m m e s i J J m m t m & & (3)
Where m i is the mass of the i th isotope in a considered medium defined as a form for the corrosion products, t is the time, is the convective term (balance between input and output) and except for nuclear reactions, which are mass production/loss within the considered medium, J m is an exchange mass rate between two different media. Seven different media are taken into account in a control volume and depicted in Figure 3 below. The transfer mechanisms, which are modelled in the mass balance equations, between all the considered media, are summarised in Figure 4. The corrosion and release processes are formulated in the mass balance equations as functions of their time production rates that now enable to describe the consequences of material surface treatments on the corrosion and release processes and, so that, on the primary circuit contamination [START_REF] Coulet | Etude et modélisation de l'influence des matériaux sur la contamination du circuit primaire des réacteurs à eau pressurisée par les produits de corrosion[END_REF]. The corrosion kinetics and the release kinetics can be measured for different alloys with different fabrication processes using, for instance, the CORELE loop [START_REF] Schindler | Stainless steel release rate evaluation in ITER operating conditions[END_REF].

Computer Aspects

Combined with a fully object oriented programming language (C ++ ), the PACTOLE V3 code has been written and designed using the Unified Method Language (UML). Thanks to its code architecture, the latest PACTOLE version is an easy evolving computer tool in which new media can be added or removed and in which new models can be tested. Moreover, the class management process, introduced in the PACTOLE code, allows a non-limited number of control volumes, chemical elements, isotopes. The only restriction comes from the memory of the computer on which the code runs and the longer run duration.

The PACTOLE V3 code version is available on UNIX or LINUX workstation and on personal computers using Windows operating system. The first simulations with PACTOLE V3.0 have shown a reasonable agreement with the on-site measurements [17].

Further developments in the PACTOLE/PACTITER code

Major improvements have been achieved in the V3.0 PACTOLE code version for the data processing architecture and numerical analysis for solving transient equations [START_REF] Marchetto | Modélisation et Simulation Numérique du Transport des Produits de Corrosion dans le Circuit Primaire des Réacteurs à Eau Pressurisée[END_REF]. This new architecture will allow the PACTOLE code of being an integration tool for the complex physics involved in contamination transfer studies. Some physical models have been already modified (among them corrosion and release rates), but there are still both existing models to improve and new models to implement. The making of a wilful work program for improving the physical models and the data is under way. The chemistry module is being improved for the PACTOLE V3.1 code. A new approach for oxide determination and equilibrium concentration calculation has been developed by CEA. This model, based on a survey of oxides thermodynamic behaviour, is aimed at predicting in a more accurate way the equilibrium concentration values. Other important steps in the PACTOLE code development for version 3.1 are scheduled within short term:

• Improvement of the corrosion/release mechanism. Based on the present empirical mathematical expression, the impact of macroscopic parameters such as solubility or temperature will be accounted for. • Addition of a specific module for shutdown. In the present code modelling, only reducing conditions can be treated: a real simulation of oxygenation phase is therefore impossible. Due, for a large part, to the lack of available data, an important effort is devoted to the data collection of oxide characteristics in oxidizing conditions. It is foreseen that the PACTITER v3.1 code development will profit by these improvements. The major needs related to the adaptation of PACTOLE V3.1 to the fusion case in general and to ITER in particular, with the aim to get PACTITER V3.1 are related to:

• the addition of Cu to the other five elements (Fe, Ni, Co, Cr and Mn);

• the consideration of the variable neutron flux distribution on the diverse FW/shield wet surfaces under neutron bombardment (two orders of magnitude between the maximum and minimum neutron flux value);

• the simulation of pulsed operation characterised by different coolant temperatures and flow rates (five main operating conditions are envisaged for ITER: burn, dwell, cold stand-by, hot stand-by and baking).

Conclusions

The development and validation work has been scheduled with the aim to have PACTITER V3.1 released within the end of 2006. The application of PACTOLE/PACTITER to the prediction of ACP for fusion reactors has been lasting for more than 10 years. The extensive work of development, qualification and validation through dedicated experiments, and application of PACTITER to ACP assessment, mainly for ITER safety analyses (release in accident conditions and occupational dose to the staff), has produced valuable results. The ultimate aim is to further reduce the uncertainties in the source terms considering the specific material and operating scenarios of ITER machine. This effort will also fulfill some of the requirements for ITER licensing in Europe.

Figure 3 -

 3 Figure 3 -Media in a Control Volume for a PACTOLE representation

Figure 4 -

 4 Figure 4 -Transfer processes between media in a control volume

Table 1 -

 1 ITER PHTS design evolution

	Year	ITER Design Evolution	ITER Thermal Power removed by PHTS [MW]	Total number of loops	divided in
	1995	Draft IDR	2325	10	4 for DIV, 4 for FW/SB, 2 for VV
					4 for DIV, 10 for
	1996	IDR	2413	20	FW/shield/IBB, 4 for
					Lim/OBB, 2 for VV
	1998	FDR	Same as IDR but using the HX tube side for secondary side
	2001	FDR	932	6	3 for FW/Sh, 1 for DIV, 2 for VV
	Note: IDR = Interim Design Report; FDR = Final Design Report; DIV = Divertor; FW/SB = First
	Wall/Shielding Blanket; VV = Vacuum Vessel; FW/shield/IBB = First Wall/Shield/InBoard Blanket; Lim/OBB
	= Limiter/OutBoard Baffle; FW/Sh = First Wall/Shield

Figure 2 -ITER Primary Heat Transfer System (FDR 1998) and PACTITER geometric model of one PHTS loop of the FW/shield/Inboard baffle

Table 4 -

 4 ITER TCWS cooling loop material average release rate

	Cooling loop type	Scenario duration	SS316L release rate	SS304L (°) release rate	Cu-alloy release rate
		[days]	[µm/year]	[µm/year]	[µm/year]
	FW/shield	3681	0.16	0.07	(*)
	Divertor	3681	0.16	0.11	2.18
	VV	3681	0.008	0.047	(*)
	(°) material in the non-irradiated zones (*) no Cu-alloy in this cooling loop

Acknowledgements

This work, supported by the European Communities under the Contract of Association between EURATOM on one side and CEA / ENEA on the other side, was carried out within the framework of the European Fusion

Development Agreement. The views and opinions expressed herein do not necessarily reflect those of the European Commission. The authors would thank V. Blet and P. Schindler for useful discussions during the preparation of the present paper.