Yohan Pillon

Helen C F Hopkins

Jérôme Munzinger

Mark W Chase

A molecular and morphological survey of generic limits of Acsmithia and Spiraeanthemum (Cunoniaceae)

Abstract─A phylogenetic analysis was conducted on the tribe Spiraeanthemeae (Cunoniaceae) to clarify relationships of the genera Acsmithia and Spiraeanthemum. Three molecular markers, one plastid region (trnL intron and trnL-trnF intergenic spacer) and two nuclear-single copy genes (ncpGS and PHYC), were sequenced for this purpose. The independent analysis of the three markers and a combined analysis all showed that the genus Acsmithia is paraphyletic since the genus Spiraeanthemum is nested within it. A morphological survey of all species in the tribe confirmed the existence of two groups within Acsmithia. One comprises the species from Australia, New Guinea, and A. densiflora from New Caledonia and is characterised by multiple ovules per carpel. The other group contains all the remaining New Caledonian species plus A. vitiensis from Fiji and is characterised by a single ovule per carpel. The study shows that characters previously used to distinguish Acsmithia and Spiraenthemum, phyllotaxy and sexual system, are homoplasious as in several other groups of Cunoniaceae. A broad circumscription of the genus Spiraeanthemum is adopted here that includes the species formerly placed in Acsmithia. Two new combinations are proposed: Spiraeanthemum collinum and Spiraeanthemum meridionale.

Spiraeanthemum austrocaledonicum is considered a synonym of Spiraeanthemum densiflorum.

Keywords─ glutamine synthetase, Melanesia, New Caledonia, nuclear-single copy gene, phyllotaxy, sexual system. Cunoniaceae (Oxalidales) are a predominantly Southern Hemisphere family of trees

and shrubs consisting of 27 genera and ca. 300 species [START_REF] Bradford | The families and genera of vascular plants[END_REF]). Molecular phylogenetic studies of the family [START_REF] Bradford | Phylogenetics and classification of Cunoniaceae (Oxalidales) using chloroplast DNA sequences and morphology[END_REF] allowed circumscription of a number of monophyletic tribes within the family for several groups of genera that had been allied previously on morphological grounds [START_REF] Hufford | A phylogenetic analysis of Cunoniaceae[END_REF]. Tribe Spiraeanthemeae was shown to be sister to the remainder of the family [START_REF] Bradford | Phylogenetics and classification of Cunoniaceae (Oxalidales) using chloroplast DNA sequences and morphology[END_REF][START_REF] Sweeney | Phylogenetic position of the New Caledonian endemic genus Hooglandia (Cunoniaceae) as determined by maximum parsimony analysis of chloroplast DNA[END_REF]) and consists of two genera : Acsmithia Hoogland and [START_REF] Hufford | A phylogenetic analysis of Cunoniaceae[END_REF], the only genus of Brunelliaceae, a closely related family within Oxalidales. Synapomorphies of Spiraeanthemeae include epidermal glands, pocket domatia along the midvein, and synchronously maturing flowers [START_REF] Bradford | Phylogenetics and classification of Cunoniaceae (Oxalidales) using chloroplast DNA sequences and morphology[END_REF]. Within Cunoniaceae, none of these characters is unique to this tribe; the combination is, however, unique.

As currently circumscribed Acsmithia, consists of 14 species from eastern Malesia, Australia, New Caledonia, and Fiji; and Spiraeanthemum comprises six species from the Bismarck Archipelago, Bougainville, Solomon Islands, Vanuatu, Fiji, and Samoa. Acsmithia, named after Albert C. Smith, an authority of the flora of Fiji, was described in 1979 by R.D.

Hoogland to accommodate species formerly included in Spiraeanthemum which have whorled leaves and hermaphroditic flowers, whereas Spiraeanthemum s.s. is characterized by opposite leaves and unisexual flowers (dioecious). Although recognition of two genera was questioned by [START_REF] Hufford | A phylogenetic analysis of Cunoniaceae[END_REF], they have been considered as distinct in all regional treatments of the family published since 1979 [START_REF] Smith | Cunoniaceae[END_REF][START_REF] Hopkins | Cunoniaceae[END_REF], in Hoogland (unpublished manuscript for the Flore de la Nouvelle-Calédonie et Dépendances) and in the generic survey of the family by [START_REF] Bradford | Phylogenetics and classification of Cunoniaceae (Oxalidales) using chloroplast DNA sequences and morphology[END_REF][START_REF] Bradford | The families and genera of vascular plants[END_REF]. As part of a study on the evolution of the family in New Caledonia and Vanuatu, a molecular phylogeny of the tribe Spiraeanthemeae in this region is presented here, along with a survey of morphological characters. We were interested in assessing the monophyly Acsmithia in New Caledonia and the biogeographical patterns of both Acsmithia and Spiraeanthemum.

MATERIAL AND METHODS

Molecular analysis─

Leaf material of Acsmithia and Spiraeanthemum was dried in the field in silica gel [START_REF] Chase | Silica gel: an ideal material for field preservation of leaf samples for DNA studies[END_REF] and DNA was extracted following a modified CTAB protocol [START_REF] Csiba | DNA extraction protocols[END_REF]. Brunellia (Brunelliaceae), Hooglandia (Cunoniaceae, unplaced to tribe) and Platylophus D. Don (Cunoniaceae, tribe Schizomerieae) were also included as outgroups. The plastid trnL-F region (trnL intron and trnL-trnF spacer) was amplified with the primers c and f from [START_REF] Taberlet | Universal primers for amplification of three noncoding regions of chloroplast DNA[END_REF] using a standard protocol (e.g. [START_REF] Pillon | Evolution and temporal diversification of western European polyploid species complexes in Dactylorhiza (Orchidaceae)[END_REF]. A portion of the chloroplast-expressed glutamine synthetase (ncpGS), a nuclear single-copy gene, was amplified using the primers 687F and 994R of [START_REF] Emshwiller | Chloroplast-expressed glutamine synthetase (ncpGS): potential utility for phylogenetic studies with an example from Oxalis (Oxalidaceae)[END_REF]. PCR reaction mix included 45 L of 2.5 mM MgCl2 Reddy PCR Master Mix 1.1× (ABgene Ltd, Surrey, U.K.), 1 L of 0.4% bovine serum albumin (BSA), 0.5 L of each primer (100 ng/L) and 3 L of template DNA. The following PCR program was used: 2 min at 94°C, 38 cycles of 1 min at 94°C, 1 min at 50°C, 1 min 30 sec at 72°C and a final extension of 5 min at 72°C. A portion of the first exon of phytochrome C (PHYC), another nuclear single-copy gene, was amplified using the primers PhyC-F and PhyC-R from [START_REF] Samuel | Molecular phylogenetics of Phyllanthaceae: evidence from plastid matK and nuclear PHYC sequences[END_REF]. The PCR reaction mix included 45 L of 1.5 mM MgCl2 Reddy PCR Master Mix 1.1× (ABgene Ltd), 1.5 L of 0.4% bovine serum albumin (BSA), 0.5 L of each primer (100 ng/L), 1 L of DMSO and 2 L of template DNA. The following PCR program was used: 3 min at 94°C, 38 cycles of 1 min at 94°C, 1 min at 50°C, 1 min at 72°C and a final extension of 10 min at 72°C. For nuclear genes, cloning for some accessions was necessary to separate alleles. Alignment and phylogenetic analyses were carried out with PAUP*4.01b10 [START_REF] Swofford | PAUP*: Phylogenetic Analysis Using Parsimony (and Other Methods) 4.0 Beta[END_REF]. To recover the most parsimonious trees, we used branch and bound searches. Robustness of tree topology was assessed using bootstrapping with 100 replicates, also with branch and bound searches. For the combined analysis, a single allele was retained for each accession; we chose the allele that appeared as the most ancestral (the one on the shortest branch). For Brunellia the sequences from two different species were combined. The list of species, vouchers and GenBank accession numbers are provided in the appendix. Sequence datasets and phylogenetic trees were deposited in Treebase (study accession number: S2119, matrix accession numbers: M4005, M4006, M4007). Morphology─Morphological characters either observed by us or mentioned in the literature as differing between Spiraeanthemum and Acsmithia or varying among Acsmithia species were scored with the aid of a binocular microscope ( 10-40 magnification) for all species in both genera in the herbaria at Kew (K) and Nouméa (NOU). The only character not scored by us was the number of ovules per carpel, for which data were taken from Hoogland (1979, unpublished manuscript, and[START_REF] Hopkins | Nomenclature and typification in Geissois (Cunoniaceae) in the South-West Pacific[END_REF][START_REF] Hopkins | Cunoniaceae[END_REF]. Literature sources used to suggest possible characters included these works by Hoogland as well as [START_REF] Bradford | Phylogenetics and classification of Cunoniaceae (Oxalidales) using chloroplast DNA sequences and morphology[END_REF], [START_REF] Bradford | The families and genera of vascular plants[END_REF], [START_REF] Dickison | Fruits and seeds of the Cunoniaceae[END_REF], [START_REF] Rutishauser | Developmental morphology of stipules and systematics of the Cunoniaceae and presumed allies. I. Taxa with interpetiolar stipules[END_REF], [START_REF] Hufford | A phylogenetic analysis of Cunoniaceae[END_REF], [START_REF] Hyland | Australian Tropical Rain Forest Trees, an interactive identification sytem[END_REF][START_REF] Hyland | Australian Tropical Rain Forest Trees, an interactive identification sytem[END_REF][START_REF] Smith | Studies of Pacific Island plants, XII, the Cunoniaceae of Fiji and Samoa[END_REF][START_REF] Smith | Cunoniaceae[END_REF].

RESULTS

Molecular analysis─

Detailed statistics for each DNA regions studied are provided in Table 1. The two nuclear-single copy genes both had a greater proportion of variable and parsimony informative characters than the plastid trnL region. The ncpGS region was particularly informative, with nearly 20% of sites variable, over ca. 1,200 base pairs. It is thus a promising tool for phylogenetic studies in Cunoniaceae and Oxalidales in general.

Analysis of trnL-F sequences (Fig. 1) indicates that the two accessions of Acsmithia densiflora from New Caledonia form a monophyletic group that is sister to a well-supported group (94 bootstrap percentage, BP) consisting of all other species of the tribe. In this group we can distinguish Spiraeanthemum s.s. as well supported (91 BP); it is represented by S. macgillivrayi (Vanuatu) and S. samoense (Samoa). The sister group of Spiraeanthemum s.s. is a moderately supported clade (67 BP) consisting of four species of New Caledonian Acsmithia: A. brongniartiana, A. elliptica, A. meridionalis, and A. pubescens (hereafter referred to as the brongniartiana group).

Analysis of ncpGS (Fig. 1) also recovered A. densiflora as sister to the remaining members of Spiraeanthemeae, and Spiraeanthemum s.s. as sister to the brongniartiana group represented by A. brongnartiana and A. pubescens. Monophyly of the brongniartiana group and of the clade Spiraeanthemum s.s.-brongniartiana group are both well supported (99 and 100 BP).

In the PHYC analysis (Fig. 1)¸ A. densiflora is again sister to the remaining members of the tribe. Spiraeanthemum macgillivrayi is sister to the brongniartiana group represented by A. brongniartiana, A. collina, A. elliptica, A. pedunculata and A. pubescens. In the combined analysis (Fig. 1), we recovered the same topology as for the three separate analyses, with a strongly supported (100 BP) Spiraeanthemeae structured as followed (A. densiflora (Spiraeanthemum s.s.; brongniartiana group)) with 100 BP for the brongniartiana group and Spiraeanthemum s.s. plus the brongnartiana group. A combined analysis including all taxa for which at least one of the loci was available was also conducted.

The topology recovered was identical to the one recovered in the reduced dataset with no missing loci, but bootstrap supports were lower, it is therefore not shown here.

Morphology─Since the phylogenetic analysis suggested that three groups of species could be recognised within Spiraeanthemum-Acsmithia (see discussion), morphology is described largely in terms of these groups. Species of Acsmithia not included in the phylogenetic analysis were assigned initially to either the brongniartiana group or the densiflora group on the basis of their ovule number. STIPULES AND APICAL BUDS─Two types of interpetiolar stipules were found, and among the species with small, linear stipules, two types of apical buds occur.

In Spiraeanthemum s.s., interpetiolar stipules are large, ovate to oblong-elliptic, and attached broadly at the base. In dormant apical buds, opposite stipules are adpressed around their margins to enclose the developing leaves; after separating, they are caducous or sometimes briefly persistent, and leave a long, curved scar.

In both the brongniartiana and densiflora groups, interpetiolar stipules are narrowly triangular to linear, with a small attachment at the base (see Rutishauser and Dickison 1989, Figs. 80-82, A. densiflora, given as A. undulata). Although stipules overtop leaves in small buds [START_REF] Rutishauser | Developmental morphology of stipules and systematics of the Cunoniaceae and presumed allies. I. Taxa with interpetiolar stipules[END_REF], in dormant apical buds they are not contiguous with one another and do not enclose the developing leaves, which are visible between them and larger; as the apical bud starts to develop, stipules are usually fugaceous, leaving a small round or elliptic scar. It appears that stipules are not smaller in these two groups only because whorled phyllotaxis means that more stipules and petioles are present at each node; their shape and persistence differs from those in Spiraeanthemum s.s.

Apical buds in both Spiraeanthemum s.s. and the densiflora group are hairy, whereas in the brongniartiana group they are glabrous and varnished except in A. pubescens, with some rare exceptions (e.g. A. pubescens: Pancher Mus. Neocal. 191, some apical buds glabrous and varnished; A. meridionalis: Bernardi 12434, apical bud hairy with some varnish).

LEAVES─These have either toothed margins and craspedodromous or semicraspedodromous venation, or entire margins with brochidodromous venation. Although distinctive patterns of venation can be picked out, some species or individuals within each group have a more generalized type of venation, so these categories are of only limited use.

Three species in Spiraeanthemum s.s. (S. serratum, S. samoensis, and S. macgillivrayi) have markedly serrate margins with numerous teeth and semicraspedodromous venation in which branches of the secondary veins reach the margin at the sinus of a tooth. The secondary veins are numerous, regularly spaced along the midrib, parallel to one another across the blade, and prominent on the abaxial surface; leaf blades are flat. In S. bougainvillense, teeth are much smaller and the pattern of secondary veins is less marked, whereas in S. graeffei and S. katakata margins can be entire or toothed, in which case teeth are small.

In the brongniartiana group, all species have entire margins and brochidodromous venation. Secondary veins are not especially numerous, but they are usually regularly spaced and parallel and either flat or prominent on the abaxial surface; leaf blades are flat or slightly bullate.

In the densiflora group, especially in New Guinea, the leaf margin is commonly entire or sometimes weakly toothed, especially in A. reticulata, and venation is brochidodromous to craspedodromous, in which case secondary veins reach the margin at the tip of a tooth. The secondary veins are typically few and irregularly spaced along the midrib, sometimes with weaker veins between them, and they are often not parallel to one another across the blade.

Veins are prominent on the abaxial surface, and leaf blades are flat to bullate. Only in A.

davidsonii are leaves sometimes strongly toothed.

INFLORESCENCES─In all species except A. davidsonii, panicles are typically axillary, and in most species they are exclusively axillary, although, especially in the densiflora group in New Guinea, median panicles are also found occasionally. Acsmithia davidsonii has predominantly median panicles.

The branching pattern within panicles changes acropetally. In general, branching initially follows the phyllotaxis of the leaves (opposite and decussate in Spiraeanthemum s.s. and whorled in Acsmithia, but see comments for densiflora group) and switches distally to become ± alternate or irregular.

Considering only axillary panicles, first branches on the main axis are produced well above the base in Spiraeanthemum s.s. and the brongniartiana group. However, in S. macgillivrayi subsp. macgillivrayi, axillary buds or panicles are occasionally accompanied by additional axillary buds (e.g. Pillon 570, Wheatley 341), and in the brongniartiana group, leaf axils occasionally contain two panicles in series (e.g. A. brongniartiana: Balansa 2303, MacKee 4432, 15327, 30991;A. meridionalis: Bernardi 12434, Pancher s.n.;A. pubescens: MacKee 37894;A. vitiensis : Horne 1104A. vitiensis : Horne , 1113)). Usually one of these panicles is much larger than the other, giving the appearance of a single panicle that is branched at the base of its main axis.

In the densiflora group (except A. davidsonii), first branches of axillary panicles are usually at or just above the base of the main axis, one on either side of it, i.e. these branches appear ± opposite (possibly by abortion of other branches), rather than whorled. More distal branching is usually whorled and then eventually alternate to irregular. In median panicles in this group, including those of A. davidsonii, first branches are well above the base.

The difference between basally branched panicles in the densiflora group and additional axillary panicles in the brongniartiana group is that in the former, first branches are ± lateral, in a plane perpendicular to that of the leaf axil, whereas in the latter additional axes arising near the base are in the same plane as the axil. However, this difference can only be seen with a binocular microscope.

This could be shown diagramatically (leaves shown as opposite for simplicity) as: densiflora group: sequence: petiole, axillary bud with lateral branches, main stem, axillary bud with lateral branches, petiole. brongniartiana group: sequence: petiole, 2 axillary buds in series, main stem, 2 axillary buds in series, petiole.

FLOWERS─Although flowers in Acsmithia are normally bisexual, some rare exceptions have been found in A. meridionalis, in MacKee 42518 and 42993, in which 5 and 15% respectively of flowers lacked carpels (Hoogland ms), as in the male flowers of Spiraeanthemum s.s. (fide [START_REF] Smith | Cunoniaceae[END_REF].

OVULES AND SEEDS─The number of ovules per carpel correlates strongly with seed morphology (one ovule per carpel and seeds with a flattened wing, or, more than one ovule per carpel and seeds ellipsoid to spindle-shaped).

Species in the brongniartiana group have one ovule per carpel, and mature seeds are ellipsoid or flattened-ellipsoid with a flat, membranous wing at one end or obliquely at one end (see Dickison 1984, Fig. 54 -A. elliptica, Fig. 55-56 -A. pubescens).

In the densiflora group, the number of ovules is usually two or four (rarely three, five or six), and mature seeds are ellipsoid with a small, thin tail at each end that is easily detached (see Dickison 1984, Fig. 57 -A. reticulata). A similar picture is seen in Spiraeanthemum s.s. in which number of ovules per carpel is two and mature seeds are spindle-shaped, ellipsoid in middle and tapering gradually towards either end (see Dickison 1984, Fig. 52 -S. katakata).

However, no real difference exists between seeds in Spiraeanthemum s.s. and the densiflora group, although tails are perhaps more ephemeral in the latter. REJECTED CHARACTERS─Gland dots on the intervenium appear to be present in all species, although they are not always easily seen in old leaves and on old specimens. Domatia occur in the angle between secondary veins and the midrib in all species, although not necessarily on all leaves or all specimens, and in a few species they are small. Some species have additional domatia in the angle between major branches of the secondary veins towards the margin. Domatia vary from simple hair-tufts to pits and pouches [START_REF] Hopkins | Cunoniaceae[END_REF], Fig. 3), and, although certain types are more common in certain species, they are too variable to be taxonomically useful. The number of calyx lobes and carpels, form of disc, and extent to which carpels are connate towards their bases all appear to show more variation within species than between them.

DISCUSSION

Separate and combined analyses of the three DNA regions all showed that two groups can clearly be distinguished within New Caledonian Acsmithia. The first consists of A. densiflora only and the second (the brongniartiana group) of the remaining species. The brongniartiana group is a well supported, and a more complete analysis (Pillon et al., in prep.) shows that it includes all New Caledonian taxa except A. densiflora and A. austrocaledonica, which is in fact conspecific with A. densiflora (see below). The brongniartiana group and Spiraeanthemum s.s. form a well-supported clade to which A. densiflora is sister.

Acsmithia densiflora can be distinguished from species of the brongniartiana group by a number of morphological features (Table 2) including number of ovules per carpel. This character was the first used in Hoogland's key to the species of Acsmithia [START_REF] Hoogland | Studies in the Cunoniaceae. II. The genera Caldcluvia, Pullea, Acsmithia and Spiraeanthemum[END_REF] and allows placement of non-New Caledonian species of Acsmithia in one or the other group, even though material was not available for molecular analysis.

Acmithia vitiensis, the only species of Acsmithia in Fiji, is morphologically similar to A. brongniartiana and like all species of the brongniartiana group has a single ovule per carpel. Acsmithia pulleana (type of Acsmithia), A. parvifolia, A. integrifolia, A. reticulata (all from New Guinea), and A. davidsonii (Australia) have several (two to four or rarely six) ovules per carpel, as in A. densiflora.

The two groups within Acsmithia also differ in other characters. Members of the densiflora group all have hairy apical buds, whereas in the brongniartiana group apical buds are glabrous and often resinous, with the notable exception of A. pubescens. Axillary panicles in the densiflora group are ramified at, or close to, the base (within the first few millimetres), whereas those in the brongniartiana group have a marked peduncle, often extending a third or more of the total length of the panicles. Seeds in the densiflora group are ellipsoid with a small tail at either end, whereas those in the brongniartiana group are flattened to ellipsoid with a membranous wing at one end.

Within the densiflora group, Acsmithia davidsonii has several characters that are unusual: leaves are sometimes strongly toothed; panicles are mostly median, and even in axillary ones, the lowest branches are above the base. However, the number of ovules, shape of the seeds, and secondary venation are all consistent with other members of the densiflora group.

The morphological survey also confirmed a character shared by the two groups within Acsmithia but not by Spiraeanthemum that was first noted briefly by [START_REF] Smith | Studies of Pacific Island plants, XII, the Cunoniaceae of Fiji and Samoa[END_REF]. In Acsmithia stipules are small and narrowly triangular, whereas in Spiraeanthemum s.s. they are larger and broadly ovate to oblong-elliptic. This second type of stipule is common, although not universal, throughout Cunoniaceae [START_REF] Rutishauser | Developmental morphology of stipules and systematics of the Cunoniaceae and presumed allies. I. Taxa with interpetiolar stipules[END_REF].

Evolution of the different characters observed can be tracked by comparing them with the topology observed in the molecular trees. The following characters are homoplasious in Spiraeanthemeae and have either evolved twice or are ancestral in the tribe but show a reversal: narrowly triangular stipules, hairy apical buds, whorled leaves, bisexual flowers, multiple ovules per carpel and spindle-shape/ellipsoid seeds. The densiflora group has a single autapomorphy, panicles branched near the base, and the clade Spiraeanthemum s.s.brongniartiana group is also only characterised by a single character, a clearly pedunculate inflorescence. Autapomorphies for Spiraeanthemum s.s. include opposite leaves, ovateelliptic stipules and dioecy. Autapomorphies for the brongniatiana group include glabrous apical buds, a single ovule per carpel, seeds with membranous wings and adaptation to ultramafic soils in New Caledonia. Future biochemical studies may provide further characters to support these groups, as bioactivity and taxonomy are well correlated within Cunoniaceae [START_REF] Pillon | Evidence for a correlation between phylogeny and bioactivity in New Caledonian Cunoniaceae and its implications for screening and conservation[END_REF] In terms of biogeography, the densiflora group is restricted to the relatively ancient landmasses of New Guinea, Australia and New Caledonia (Fig. 2) and has a distribution comparable to Dubouzetia Brong. & Gris (Elaeocarpaceae, Coode 2004) or Sphenostemon Baill. (Sphenostemonaceae, Jérémie 1997). The brongniartiana group, restricted to New Caledonia and Fiji (Fig. 2), can be compared to Acmopyle Pilg. (Podocarpaceae, de Laubenfels 1996) and can be considered as a typical eastern Melanesia endemic, like Cyphosperma H. Wendl. (Arecaceae, [START_REF] Dowe | A taxonomic account of Arecaceae in Vanuatu, with descriptions of three new species[END_REF] or Geissois Labill. s.s. (Cunoniaceae, Hopkins 2006). Spiraeanthemum s.s. has a relatively large distribution covering most islands of the Melanesian arc from New Britain to Fiji and extending to western Polynesia (Samoa) but excluding New Caledonia ( Fig. 2): most of these islands have a volcanic origin. Several palm genera show similar distributions, e.g. Metroxylon Rottb.¸Clinostigma H. Wendl., and Physokentia Becc. [START_REF] Dowe | A taxonomic account of Arecaceae in Vanuatu, with descriptions of three new species[END_REF]. Spiraeanthemum s.s. reaches its western limit in the Bismarck Archipelago and the Solomon Islands. Another group of Cunoniaceae that reaches its distributional limit here is the Pacific section of Weinmannia, sect. Leiospermum (D. Don) Engl., but its distribution is wider than that of Spiraeanthemum s.s. as it extends eastwards to French Polynesia and southwards to New Caledonia and New Zealand [START_REF] Hopkins | A revision of Weinmannia (Cunoniaceae) in Malesia and the Pacific. 1. Introduction and an account of the species of Western Malesia, the Lesser Sunda Islands and the Moluccas[END_REF].

Acsmithia was created to accommodate species of Spiraeanthemum with whorled leaves and hermaphroditic flowers, as opposed to those with opposite leaves and a dioecious sexual system in Spiraeanthemum s.s. However, it seems that whorled leaves and hermaphroditism are ancestral characters within the tribe Spiraeanthemeae, and transitions between opposite and whorled leaves have occurred several times in the course of the evolution of Cunoniaceae. For instance, in the New Caledonian endemic genus Pancheria Brong. & Gris, all species have whorled leaves with the exception of P. confusa Guillaumin, which has opposite ones. Conversely, all species of Codia J.R. Forst. & G. Forst., which is also endemic to New Caledonia, usually have opposite leaves except for C. albifrons (Brongn.

ex Schinz & Guillaumin) Baker f. and C. triverticillata H.C. Hopkins & Pillon in which they are whorled [START_REF] Hopkins | Four new species in the endemic genus Codia (Cunoniaceae) from New Caledonia[END_REF]. Whorled leaves are clearly derived when mapped onto a phylogenetic tree of Codia (Pillon et al., unpublished), and they have been recorded as a rare occurrence in most taxa in this genus. In Pullea glabra Schltr. in New Guinea, both opposite and verticillate leaves occur regularly within a single species, in var. glabra and var. verticillata Hoogland respectively. Teratological cases are not uncommon in Cunoniaceae, and for example in Cunonia L., in which all species normally have opposite leaves, twigs with whorled leaves have been observed on a potted plant of C. pterophylla Schltr. (Pillon, pers. obs.). Sexual systems are also labile characters that can show great variability, as seen for instance in Weinmannia L. [START_REF] Hopkins | A revision of Weinmannia (Cunoniaceae) in Malesia and the Pacific. 1. Introduction and an account of the species of Western Malesia, the Lesser Sunda Islands and the Moluccas[END_REF], and, furthermore, sexual systems are capable of evolving quickly (e.g. [START_REF] Sakai | Phylogenetic perspectives on the evolution of dioecy: adaptive radiation in the endemic Hawaiian genera Schiedea and Alsinodendron (Caryophyllaceae: Alsinoideae)[END_REF]. Thus phyllotaxis and sexual system, even in combination, can be misleading characters in classification of Cunoniaceae.

The paraphyly of Acsmithia implies that taxonomic changes need to be made. Two alternatives are possible: either we could treat tribe Spiraeanthemeae as a single genus, Spiraeanthemum s.l., or as three distinct genera: Spiraeanthemum s.s., a narrowly defined Acsmithia comprising the densiflora group, and an undescribed genus which would contain all New Caledonian and Fijian species except for A. densiflora. The first alternative is preferred for several reasons. Firstly, the densiflora and brongniartiana groups are morphologically similar, and differences between them are in characters that are difficult to observe; splitting Acsmithia into two would not aid generic identification by a non-specialist nor by anyone without a microscope. Secondly, although Spiraeanthemum s.s. and the brongniartiana group each have several synapomophies, the densiflora group has only one (axillary panicle

  Spiraeanthemum A. Gray. The members of this tribe are unique in the family in being apocarpous (but see the recently described Hooglandia McPherson & Lowry which is monomerous, McPherson and Lowry 2004) and a close relationship has been suggested with Brunellia Ruiz & Pavón

Australia

  FIG 1. Strict consensus trees of most parsimonious trees resulting from phylogenetic

TABLE 1 .

 1 Statistics for each DNA sequences used in the phylogenetic analysis of the tribe Spiraeanthemeae.

	trnL	PHYC	ncpGS	combined

TABLE 2 .

 2 Comparative morphology between the different species and species group of Acsmithia and Spiraeanthemum. Rare occurrences are given in brackets. A=axillary, M=median. ?=see text.

										Number
				Number		position	branching	additional		ovules
				of leaves		of	in axillary	axillary	sexual	per
	species	stipules	apical bud	per node leaf margin	panicles	panicles	buds	expression	carpel	seed shape
	densiflora group
		narrowly
	densiflora	triangular	hairy	3	entire	A	near base no	bisexual	4 (to 6)	ellipsoid with tails
		narrowly
	integrifolia	triangular	hairy	3 or 4	entire	A + M	near base no	bisexual	4	not seen
		narrowly
	parvifolia	triangular	hairy	3 (4)	entire	A + M	near base no	bisexual	2	ellipsoid with tails
		narrowly
	pulleana	triangular	hairy	3 or 4	mostly entire	A (M)	near base no	bisexual	2 (3)	ellipsoid with tails
		narrowly			toothed to ±
	reticulata	triangular	hairy	3 or 4	entire	A	near base no	bisexual	2 (3)	ellipsoid with tails
		narrowly					above
	davidsonii	triangular	hairy	3 (4)	toothed	M (A)	base	no	bisexual	2 to 4	ellipsoid with tails
	brongniartiana group
		narrowly					above
	brongniartiana	triangular	glabrous/varnished 3 (4)	entire	A	base	yes?	bisexual	1	with membranous wing
		narrowly					above
	collina	triangular	glabrous/varnished 3 to 5	entire	A	base	no	bisexual	1	with membranous wing
		narrowly					above
	elliptica	triangular	glabrous/varnished 4	entire	A	base	no	bisexual	1	with membranous wing
		narrowly					above
	meridionalis	triangular	glabrous/varnished 4 (5)	entire	A	base	yes?	bisexual	1	with membranous wing
		narrowly					above
	pedunculata	triangular	glabrous/varnished 4	entire	A	base	no	bisexual	1	with membranous wing
		narrowly					above
	pubescens	triangular	hairy	4	entire	A	base	no	bisexual	1	with membranous wing
		narrowly					above
	vitiensis	triangular	glabrous/varnished 3 or 4 (5) entire	A	base	yes?	bisexual	1	with membranous wing
	Spiraeanthemum s.s.

TABLE 3 .

 3 List of taxa currently accepted in the genus Spiraeanthemum. Species belonging to Spiraeanthemum s.s., the densiflorum and brongniartianum group are marked with a S, D and B respectively.


Yohan Pillon

Jérôme Munzinger

Hamid Amir

Michel Lebrun

Ultramafic soils and species sorting in the flora of New Caledonia

Keywords: island biogeography, phylogenetic clustering, phylogenetic community structure, phylogenetic niche conservatism, serpentine, species selection

Running headline: Species sorting in the flora of New Caledonia 6. Synthesis. This study supports that beyond dispersal, ecology is an important factor in determining the success of different plant groups to colonize an island. In the case of New Caledonia, exaptation to ultramafic soils is likely a primary cause of ecological filtering.

SUMMARY

1. Island floras are generally characterized by disharmony due to uneven representations of plant groups. This has usually been explained by the unequal capacity of plants to disperse and colonize islands, but it has been suggested that the ecology of the recipient islands may also be important.

2. We investigated the phylogenetic structure of the flora of New Caledonia, a biodiversity hotspot in the South-West Pacific, to examine the phylogenetic pattern of a disharmonic flora and to test two competing hypotheses: disharmony due to uneven dispersability versus disharmony due to uneven ability to adapt to the ultramafic soils that are widespread on the archipelago and constrain plant growth.

3. Dispersal did not seem to be a significant factor as plant families with low levels of endemism and thus high effective dispersal capacities tended to be under-represented.

4. We identified several clusters of over-represented families, such as the COM clade (Celastrales, Oxalidales and Malpighiales), in which nickel hyperaccumulation (a syndrome associated with ultramafic soils) was particularly common. We indeed found a positive association between hyperaccumulation and over-representation. Therefore, ultramafic soils may have been a major force for species sorting in the flora of New Caledonia.

5. Because we think that most colonizers were naïve to ultramafic soils before they reached the archipelago, the over-represented groups in New Caledonia may have possessed an exaptation to ultramafic soils, which has facilitated their settlement and diversification.

INTRODUCTION

"… the overriding role that the ecology of the recipient island plays in dispersal cannot be strongly enough stressed." (Carlquist 1974, p. 81) Island biotas have long inspired evolutionary biologists, from the conception of the theory of evolution by Darwin to the now common reference to islands as "evolutionary laboratories" [START_REF] Carlquist | Island Biology[END_REF]. They have often served as models for the study of adaptive radiation [START_REF] Givnish | Molecular Evolution and Adaptive Radiation[END_REF][START_REF] Schluter | The ecology of adaptive radiation[END_REF], and more recently for sympatric speciation [START_REF] Savolainen | Sympatric speciation in palms on an oceanic island[END_REF]. [START_REF] Macarthur | An equilibrium theory of insular zoogeography[END_REF] have offered a simple theory to explain the formation of island biotas. Later extended by Hubbell's neutral theory (2001), insular biodiversity is seen as an equilibrium between immigration, extinction, and local speciation. The composition of an insular biota may be disharmonic (Carlquist 1974, p.6) due to the over-or under-representation of particular groups. Disharmony of island biotas has generally been attributed to variation in dispersal capacity among groups, but local ecological conditions may be an alternative explanation.

Phylogenetic studies in various plant groups have recently revealed surprising cases of long-distance dispersal between land masses that were distant and/or non-contiguous, but which nevertheless offered similar ecological conditions: dispersal of Ourisia from the Andes to the temperate flora of New Zealand [START_REF] Meudt | The biogeography of the austral, subalpine genus Ourisia (Plantaginaceae) based on molecular phylogenetic evidence: South American origin and dispersal to New Zealand and Tasmania[END_REF], dispersal of Carex between poles [START_REF] Vollan | Genetic variation, taxonomy and mountain-hopping of four bipolar Carex species (Cyperaceae) analysed by AFLP fingerprinting[END_REF], and dispersal of Iridaceae, Orchidaceae, Poaceae and Restionaceae from the Cape Province to Eastern African Mountains [START_REF] Galley | The Cape element in the Afrotemperate flora: from Cape to Cairo[END_REF]. Using 45 groups of vascular plants of the southern Hemisphere, [START_REF] Crisp | Phylogenetic biome conservatism on a global scale[END_REF] found evidence for phylogenetic biome conservatism, which is a tendency for plant lineages to occupy the same biome after transoceanic dispersal. Such study supports the paradigm suggested by [START_REF] Donoghue | A phylogenetic perspective on the distribution of plant diversity[END_REF] that "It's easier to move than to evolve." Therefore, in the process of the formation of an island flora, rare immigrants that arrive from regions with ecological conditions similar to those present on the island may have a greater success in colonization relative to more common immigrants with poorly matched requirements.

New Caledonia, an archipelago in the south-west Pacific, represents an excellent model for investigations of island biogeography. This biodiversity hotspot [START_REF] Myers | Biodiversity hotspots for conservation priorities[END_REF] is ecologically unique because of the abundance on the main island of ultramafic soils [START_REF] Proctor | Vegetation and soil and plant chemistry on ultramafic rocks in the tropical Far East[END_REF], which support an unusual flora [START_REF] Jaffré | The relationship between ecological diversity and floristic diversity in New Caledonia[END_REF][START_REF] Morat | Our knowledge of the flora of New Caledonia: endemism and diversity in relation to vegetation types and substrates[END_REF]. Because of the unusual ecological conditions of this archipelago, the impact of the local ecology on the composition of this island flora is more likely to be detected.

Relatively few studies have considered ecology and historical biogeography of islands simultaneously [START_REF] Wiens | Historical biogeography, ecology and species richness[END_REF]). Here we investigate the phylogenetic structure [START_REF] Webb | Exploring the phylogenetic structure of ecological communities: an example for rain forest trees[END_REF] of the entire flora of New Caledonia to search for phylogenetic patterns in its disharmony and to identify clades that are over-or under-represented. We then address factors that may be responsible for species sorting [START_REF] Vrba | What is species selection?[END_REF][START_REF] Vrba | The hierarchical expansion of sorting and selection: sorting and selection cannot be equated[END_REF], also referred to in a broader sense as species selection [START_REF] Jablonski | Species selection: theory and data[END_REF][START_REF] Rabovsky | Reinventing species selection with molecular phylogenies[END_REF] or selective immigration in the context of an island [START_REF] Lomolino | Investigation causality of nestedness of insular communities: selective immigration or extinction[END_REF]. In this study we aimed to investigate the relative roles of dispersal and prevailing ecological conditions in the origin of disharmony in this archipelago. Just as fire may be an important cause for phylogenetic clustering in a Mediterranean flora [START_REF] Verdú | Fire drives phylogenetic clustering in Mediterranean Basin woody plant communities[END_REF], we aimed to test if ultramafic soils have constrained immigration to New Caledonia.

MATERIAL and METHODS

MODEL SYSTEM: NEW CALEDONIA Located in the south-west Pacific, with an area of c. 19 000 km 2 , New Caledonia comprises one main island (c. 16 000 km 2 ) surrounded by several smaller ones and is situated c. 1500 km east of Australia. It was once part of Gondwana and split from Australia c. 80 million years ago [START_REF] Mcloughlin | The breakup history of Gondwana and its impact on pre-Cenozoic floristic provincialism[END_REF]. However, the deep sea deposits of the early tertiary indicate that the island was well under sea level for a long time, from the late Cretaceous to the early Eocene [START_REF] Aitchison | Eocene arc-continent collision in New Caledonia and implications for regional southwest Pacific tectonic evolution[END_REF][START_REF] Picard | L'archipel néo-calédonien, 300 millions d'années pour assembler les pièces d'un puzzle géologique[END_REF][START_REF] Pelletier | Geology of the New Caledonia region and its implications for the study of the New Caledonian biodiversity[END_REF]). The island emerged in the middle-late Eocene (c. 35-45 million years ago), when the ophiolitic nappe covered the entire island [START_REF] Pelletier | Geology of the New Caledonia region and its implications for the study of the New Caledonian biodiversity[END_REF]. Thus far, no evidence has been found to support the continuous persistence of a biota somewhere on the island or on putative islands in the vicinity, which could have served as refugia [START_REF] Grandcolas | New Caledonia: a very old Darwinian island?[END_REF]. This implies that all plant species colonized the newly emerged island through long-distance dispersal from surrounding sources [START_REF] Thorne | Floristics relationships of New Caledonia[END_REF][START_REF] Pelletier | Geology of the New Caledonia region and its implications for the study of the New Caledonian biodiversity[END_REF][START_REF] Grandcolas | New Caledonia: a very old Darwinian island?[END_REF]. All results from molecular clock analyses on New Caledonian radiation are consistent with a post-emersion origin of the New Caledonian biota [START_REF] Murienne | Evolution on a shaky piece of Gondwana: is local endemism recent in New Caledonia?[END_REF][START_REF] Grandcolas | New Caledonia: a very old Darwinian island?[END_REF].

The most likely origin of New Caledonian lineages is Australia, firstly because of its proximity, but also because it is able to offer high numbers of propagules due to its large size.

Strong affinities have indeed been found between the two floras [START_REF] Morat | Our knowledge of the flora of New Caledonia: endemism and diversity in relation to vegetation types and substrates[END_REF]) and confirmed by phylogenetic analysis of families such as the Cunoniaceae [START_REF] Bradford | Phylogenetics and classification of Cunoniaceae (Oxalidales) using chloroplast DNA sequences and morphology[END_REF] or Sapotaceae [START_REF] Bartish | Phylogenetic relationships among New Caledonian Sapotaceae (Ericales): molecular evidence for generic polyphyly and repeated dispersal[END_REF]. Beyond their similarities, some striking differences are also observed such as the absence or poor representation in New Caledonia of some genera that are extremely diverse and abundant in Australia (e.g. Acacia, Eucalyptus). Disharmony of the New Caledonian flora is particularly striking when comparing two families such as Cunoniaceae and Asteraceae (Fig. 1).

The most common explanation for disharmony in island biota is the different capacity of groups to disperse and colonize islands. However, long-distance dispersal is a rare phenomenon which is difficult to observe [START_REF] Nathan | Mechanisms of long-distance seed dispersal[END_REF] and has generally been estimated indirectly [START_REF] Ouborg | Population genetics, molecular markers and the study of dispersal in plants[END_REF]. Thus, to measure dispersability, we chose to use 'non-endemism' as a surrogate. Species that are indigenous, but not endemic, to an island are likely to have arrived at or dispersed out of this island recently, or recent gene flow has occurred preventing genetic drift and speciation. Thus, groups that disperse easily are expected to have a lower level of endemism and a greater number of non-endemic species. This has indeed been observed in the New Zealand flora [START_REF] Mcglone | Endemism, species selection and the origin and distribution of the vascular plant flora of New Zealand[END_REF], where groups with small propagules (minute seeds and spores) such as orchids and ferns have a lower level of endemism.

The major ecological feature of New Caledonia is the abundance of ultramafic soils, which have several characteristics that are limiting for plant growth: low fertility (low N, P, K), high Mg:Ca ratio, high concentrations of heavy metals (Co, Cr, Ni, etc.), and low waterholding capacity; in addition, fire is more frequent in vegetation on such substrates [START_REF] Proctor | Vegetation and soil and plant chemistry on ultramafic rocks in the tropical Far East[END_REF]. Ultramafic soils currently cover one -third of the surface of the main island, but likely covered a significant majority of the island surface when it emerged in the Tertiary [START_REF] Pelletier | Geology of the New Caledonia region and its implications for the study of the New Caledonian biodiversity[END_REF]). These soils clearly constrain the distribution of plant species within the archipelago [START_REF] Jaffré | Changements dans la végétation de la Nouvelle-Calédonie au cours du Tertiaire : la végétation et la flore des roches ultrabasiques[END_REF][START_REF] Morat | Our knowledge of the flora of New Caledonia: endemism and diversity in relation to vegetation types and substrates[END_REF] as well as their genetic structure (Pillon et al. 2009a; see also [START_REF] Jourand | Ultramafic soils from New Caledonia structure Pisolithus albus in ecotype[END_REF] for an example in fungi), and the peculiar flora of New Caledonia suggests that not all plant groups are equally able to develop in such environment. Some plants growing on ultramafic soils have developed particular syndromes: they accumulate in their tissue heavy metals such as Ni or Mn in anomalous concentration (e.g. [START_REF] Jaffré | Sebertia acuminata: a hyperaccumulator of nickel from New Caledonia[END_REF]. New Caledonia has the largest concentration of heavy metal hyperaccumulators, comparable to that of Cuba [START_REF] Brooks | Geobotany and hyperaccumulators. Plants that hyperaccumulate heavy metals[END_REF].

DATA

To test for a phylogenetic pattern of over-representation in the flora of New Caledonia, we considered all of the world's angiosperm families (N=440) and took their total number of species worldwide from [START_REF] Stevens | Angiosperm phylogeny website. Version 9[END_REF] and number of indigenous and endemic species in New Caledonia from [START_REF] Jaffré | Composition and characterisation of the native flora of New Caledonia[END_REF], and total number of indigenous species in Australia from [START_REF] Hnatiuk | Census of Australian vascular plants[END_REF]. Although the latter two lists are considered outdated, they are not expected to bias estimations of disharmony between the Australian and the New Caledonia flora. In addition to indigenous species, endemics species were used as they are expected to be free of cryptogenic species (e.g. species of doubtful indigenous status that may actually be ancient human introduction, intentional or not); the latter may not be randomly distributed among families. We used the complete family tree of [START_REF] Davies | Darwin's abominable mystery: insights from a supertree of the angiopserms[END_REF], available from the Phylomatic website [START_REF] Webb | Phylomatic: tree assembly for applied phylogenetics[END_REF]. The number of nickelhyperaccumulating species within each family was established from the list of [START_REF] Brooks | Geobotany and hyperaccumulators. Plants that hyperaccumulate heavy metals[END_REF].

COMPARATIVE ANALYSES

To assess if one family was more or less represented than expected in New Caledonia, we compared separately the observed number of indigenous and endemic species with the expected numbers of each if the flora of New Caledonia were a random set of the Australian flora (null model). For instance, if 10% of the Australian angiosperms are orchids, 10% of indigenous and endemic New Caledonian angiosperms should be orchids. This null model therefore assumes equal chance of plants to disperse and to settle in New Caledonia, i.e. equal dispersal capacities and no ecological filtering. We used a normal distribution to detect families that significantly deviated from this null model and were therefore under-or overrepresented in New Caledonia.

To test for phylogenetic correlation with over-representation we calculated for each family the following representation index:

           1 1 ln exp N N R obs
where Nobs is the number of indigenous or endemic species observed in New Caledonia, and Nexp is the number of indigenous or endemic species expected in New Caledonia under a null model. We searched for phylogenetic signal in the variation of the representation index by using Phylocom [START_REF] Webb | Phylocom: software for the analysis of phylogenetic community structure and trait evolution[END_REF]). Nodes which have significantly higher or lower means of the representation index (Tmn, mean value of trait across all descendant terminal taxa) than expected were identified by comparison with 1000 randomizations of the index value across the tips of the tree (threshold 5%) and mapped onto a phylogenetic tree using Dendroscope [START_REF] Huson | Dendroscope: an interactive viewer for large phylogenetic trees[END_REF]). K statistics of [START_REF] Blomberg | Testing for phylogenetic signal in comparative data: behavioral traits are more labile[END_REF], which measures the amount of phylogenetic signal across traits and trees, were calculated with the Picante package [START_REF] Kembel | Picante: R tools for integrating phylogenies and ecology[END_REF] in R (R Development Core Team 2008). Although values of K above 1 are expected for a character evolving in a Brownian motion model of evolution on a phylogenetic tree, this is rarely observed in real data [START_REF] Blomberg | Testing for phylogenetic signal in comparative data: behavioral traits are more labile[END_REF]. Different trait types exhibit different values in empirical studies, which rank from highest to lowest K values as follows: body size, morphology, life history, physiology and behaviour [START_REF] Blomberg | Testing for phylogenetic signal in comparative data: behavioral traits are more labile[END_REF]. To test the impact of dispersability on disharmony, we calculated the correlation coefficient between the representation index for all families that are represented in New Caledonia with the proportion of endemic species and with the number of non-endemic species in this flora.

To test for the impact of ultramafic soils on disharmony, we tested whether there was an association between significant over-representation and a hyperaccumulation index calculated for each family and defined as follows:

           1 1 ln exp Ni Ni H obs
where Niobs is the number of nickel-hyperaccumulating species recorded in a given family according to [START_REF] Brooks | Geobotany and hyperaccumulators. Plants that hyperaccumulate heavy metals[END_REF], and Niexp is the number of nickel-hyperaccumulating species expected if they were randomly distributed across angiosperms (species evenly distributed among families according to their total species number). We calculated phylogenetic independent contrasts [START_REF] Felsenstein | Phylogenies and the comparative method[END_REF] between the representation index R and the hyperaccumulation index H using the PDAP Package [START_REF] Midford | PDAP Package of Mesquite[END_REF]) implemented within Mesquite [START_REF] Maddison | Mesquite: a modular system for evolutionary analysis[END_REF].

RESULTS

Families that have a number of indigenous species that is significantly higher or lower than expected under the null model are listed in Supporting Information (Appendix S1) and mapped onto a phylogenetic tree in Fig. 2, which also shows nodes with significant deviation from random in the value of the mean representation index. Results obtained with endemic species are very similar and are shown in the Supporting Information (Appendix S2), where patterns of disharmony of the New Caledonian flora in comparison to the entire world flora are also shown.

There is an overall congruence between nodes with mean representation values that were significantly higher than expected by chance and significantly over-represented families which tend to occur in the same clade. For instance, Laurales (Magnoliids) and seven orders within Eudicots, including Ericales, Apiales, Gentianales, Sapindales and the COM clade (Celastrales, Oxalidales and Malpighiales, [START_REF] Zhu | Mitochondrial matR sequences help to resolve deep phylogenetic relationships in rosids[END_REF], contain altogether 63% of the 57 nodes with mean representation values that were significantly higher than expected by chance and 52% of the 71 over-represented families. Although these groups encompass altogether only 16% of the species of the angiosperms of Australia, they contain 51% and 60% of indigenous and endemic species of New Caledonia. Ninety-four percent of the 53 nodes with mean representation values that were significantly lower than expected by chance and 66% of the 47 significantly under-represented families were found in Monocots and in four orders of Eudicots: Caryophyllales, Asterales, Lamiales and Fabales. The proportions of Monocots in the Australian flora and the indigenous and endemic flora of New Caledonia are 21%, 18% and 11%, respectively. Caryophyllales, Fabales, Asterales and Lamiales contain 34% of the Australian flora but only 10% and 7%, respectively, of the indigenous and endemic flora of New Caledonia. The nodes for which the divergence contributed most to overall variation in representation index for endemic and indigenous species are given in Table 1.

K statistics of phylogenetic signal were equal to 0.564 and 0.579 for the representation index for indigenous and endemic species, respectively, and 0.525 for the occurrence of nickel-hyperaccumulating species. These figures are comparable to those observed in a wide range of life history and physiological traits and higher than those observed for behaviour traits considered as most labile [START_REF] Blomberg | Testing for phylogenetic signal in comparative data: behavioral traits are more labile[END_REF]

2003).

A possible explanation for this phylogenetic pattern is the importance of biogeography. New Caledonia could be the cradle of over-represented groups, thus explaining their abundance there. However, as the island was sunk into deep water in the Tertiary [START_REF] Pelletier | Geology of the New Caledonia region and its implications for the study of the New Caledonian biodiversity[END_REF]), its biota could not have occurred there prior to the emersion c. 35-45 million years ago, and most angiosperms families are older than this. Even assuming a vicariant origin of the New Caledonian flora (a hypothesis less and less supported, [START_REF] Grandcolas | New Caledonia: a very old Darwinian island?[END_REF]), which would be c. 80 million years, corresponding to the split period between New Caledonia and Australia [START_REF] Mcloughlin | The breakup history of Gondwana and its impact on pre-Cenozoic floristic provincialism[END_REF], the overall biota of New Caledonia is too young to be the birth place of entire orders. Thus the over-representation of large and ancient clades in New Caledonia can not be explained by an origin within the archipelago.

Areas other than Australia may have served as sources of plant lineages in New Caledonia, particularly New Guinea, but probably to a lesser extent [START_REF] Morat | Our knowledge of the flora of New Caledonia: endemism and diversity in relation to vegetation types and substrates[END_REF][START_REF] Marsch | Large-scale patterns of plant diversity and conservation priorities in South-East Asia[END_REF]. Furthermore, when comparing the New Caledonian flora with the world flora, we identified the same large clades that were under-represented (Asterales, Fabales, Lamiales, Asterales and Monocots) or over-represented (COM clade, Apiales, Ericales, Gentianales, Sapindales, Appendix S2). Thus the disharmony observed in New Caledonia is grossly the same whether we postulate Australia as the sole source of plant lineage or the entire world flora. The comparison with the world flora only shows a lower phylogenetic signal in overand under-represented families that appear less clustered because of obvious biogeographical constraints. For example, Brassicaceae seem under-represented, but this family is mostly diversified in temperate areas of the northern hemisphere, whereas Myrtaceae and Proteaceae seem over-represented, but these two families are well -diversified in Australia. Biogeography may thus explain the over-or under-representation of some families, but not of larger clades.

Therefore, we think that the phylogenetic pattern observed in the disharmony of the New Caledonian flora is explained by variation among plant lineages in their capacities to disperse, settle, maintain and diversify in New Caledonia, i.e. species sorting [START_REF] Vrba | What is species selection?[END_REF][START_REF] Vrba | The hierarchical expansion of sorting and selection: sorting and selection cannot be equated[END_REF]).

POTENTIAL CAUSES OF SPECIES SORTING

Dispersal

The most represented groups in New Caledonia might be characterized by a highdispersability that allowed them to colonize the island first and more often. Plant groups with great capacity of dispersal show lower levels of endemism in the New Zealand flora [START_REF] Mcglone | Endemism, species selection and the origin and distribution of the vascular plant flora of New Zealand[END_REF]. Ferns and angiosperm families with small seeds, such as Asteraceae, Poaceae and Orchidaceae [START_REF] Harper | The shape and sizes of seeds[END_REF], have indeed lower levels of endemism than other families and are represented by numerous non-endemic genera, suggesting a large number of colonization events. Nevertheless, the first two families are significantly underrepresented in New Caledonia. When comparing Asteraceae and Cunoniaceae (Fig. 1), the first family is characterized by many more events of colonization but is nevertheless less diversified on the island. More generally, endemism was positively correlated with representation in the New Caledonian flora, suggesting that dispersal may not be the major factor explaining its disharmony.

Determining the role of dispersal in the making of the New Caledonian biota is a difficult issue to tackle. According to [START_REF] Carlquist | Island Biology[END_REF], the major means of colonization of remote islands are: air flotation, dispersal by birds (internally or externally) and oceanic drift (by flotation or on raft). The first would favor small seeds, whereas large seeds are more often associated with zoochory or water-dispersed mangroves (Moles et al. 2005b). Furthermore, seed mass is often strongly correlated with ecology, especially habit (Moles et al. 2005a;Moles et al. 2005b), and therefore confounding effects can be expected for teasing apart dispersal and niche conservatism effects. In addition, island plants may evolve towards reduced dispersability, and seed size can evolved drastically after colonization [START_REF] Carlquist | Island Biology[END_REF]. The most diverse families in New Caledonia, Myrtaceae and Rubiaceae, are both wellrepresented by species with small wind-dispersed seeds and large fleshy fruits. Molecular phylogenies have shown that some genera with wind-dispersed seeds such as Metrosideros (Myrtaceae, [START_REF] Wright | Phylogenetic analyses of New Caledonian Metrosideros and Carpolepis (Myrtaceae) for nrDNA (ITS) sequences[END_REF] or Spiraeanthemum (Cunoniaceae, Pillon et al. 2009b) as well as genera with large fleshy fruits such as Syzygium (Myrtaceae, [START_REF] Biffin | Molecular systematics of Syzygium and allied genera (Myrtaceae): evidence from the chloroplast genome[END_REF], Diospyros (Ebenaceae, [START_REF] Duangjai | A multi-locus plastid phylogenetic analysis of the pantropical genus Diospyros (Ebenaceae), with an emphasis on the radiation and biogeographic origins of the New Caledonian endemic species[END_REF] or Planchonella (Sapotaceae, [START_REF] Swenson | Molecular phylogeny of Planchonella (Sapotaceae) and eight new species from New Caledonia[END_REF]) colonized the island on multiple occasions. Further data are needed on seed and fruit characteristics in New Caledonian plants, particularly for the many small families restricted or mostly found on this archipelago, and phylogenetic analyses are needed to determine the exact number of colonization events of each family. However, our observation do not support a major role for dispersal in the disharmony of the New Caledonian flora, and this may be because of the relatively ancient age of its biota that allowed even the least likely events of dispersal to happen and because other forces may be operating.

The influence of ultramafic soils

As New Caledonia emerged in the Tertiary, the island was largely covered by the ophiolotic nappe [START_REF] Pelletier | Geology of the New Caledonia region and its implications for the study of the New Caledonian biodiversity[END_REF]. The weathering of this geological assemblage mostly composed of peridotites resulted in the formation of ultramafic soils ("serpentine"), which are challenging for plant growth [START_REF] Proctor | Vegetation and soil and plant chemistry on ultramafic rocks in the tropical Far East[END_REF]. Thus, colonizing plants had to face the double challenge to first disperse over long distances and then to be able to grow on the ultramafic soils of the island. One can expect that this represented two major obstacles for successful immigrants.

However, it is possible that some species may readily have a higher potential to adapt to such soils, as previously suggested by [START_REF] Kruckeberg | An essay: the stimulus of unusual geologies for plant speciation[END_REF]: "There is substantial evidence that populations have both the potential─by being preadapted─to tolerate unusual edaphic situations and often do become tolerant to them." This was also suggested by [START_REF] Jaffré | Changements dans la végétation de la Nouvelle-Calédonie au cours du Tertiaire : la végétation et la flore des roches ultrabasiques[END_REF] for New Caledonian plants.

Nickel hyperaccumulation may be seen as a symptom of such groups that are preadapted to ultramafic soils, and this could explain the positive and significant association between hyperaccumlation and over-representation in the New Caledonian flora. Two angiosperm genera, Phyllanthus s.l. including Glochidion (Phyllanthaceae, [START_REF] Kathriarachchi | Phylogenetics of tribe Phyllantheae (Phyllanthaceae; Euphorbiaceae sensu lato) based on nrITS and plastid matK DNA sequence data[END_REF] and Psychotria (Rubiaceae), contain a large number of nickel-hyperaccumalating species (24 and 32, respectively) distributed across several continents [START_REF] Reeves | Tropical hyperaccumulators of metals and their potential for phyotoextraction[END_REF]. These two genera are actually the two most diverse genera of the New Caledonian flora and belong to groups that are over-represented: Malpighiales and Gentianales. Although both genera are large with more than 1 000 species, most of the families of comparable size do not have as many nickel-hyperaccumulating species. Nickel-hyperaccumulating Phyllanthus s.l. species are known from Cuba, New Caledonia, Dominican Republic, Puerto Rico, Indonesia and the Philippines [START_REF] Reeves | Tropical hyperaccumulators of metals and their potential for phyotoextraction[END_REF]. Cuban and New Caledonian Phyllanthus are not closely related [START_REF] Kathriarachchi | Phylogenetics of tribe Phyllantheae (Phyllanthaceae; Euphorbiaceae sensu lato) based on nrITS and plastid matK DNA sequence data[END_REF], so the hyperaccumulation trait evolved at least twice. The broad geographic distribution of the genus suggests a much larger number of appearances of this characteristic. Similarly, nickel-hyperaccumulating species of Psychotria are known from Cuba, Puerto Rico, Australia and New Caledonia, and multiple evolution of the trait is likely.

Thus, these genera seem more efficient at adapting to ultramafic soils. Among the overrepresented clades in New Caledonia, nickel-hyperaccumulating plants are known from the three orders Celastrales, Oxalidales and Malpighiales of the COM clade, Sapindales, Gentianales, Ericales, but have not so far been found in Apiales and Laurales. The COM clade itself contains 42% of the angiosperm species known to hyperaccumulate nickel according to [START_REF] Brooks | Geobotany and hyperaccumulators. Plants that hyperaccumulate heavy metals[END_REF]. In the Asteraceae and Fabaceae, two under-represented families, the numbers of hyperaccumulating species are rather modest, considering the large species numbers in these families. Also, nickel hyperaccumulation should not be considered as a necessary condition to ultramafic soils adaptation ; it is rather a possible symptom of adaptation to ultramafic soils. Most adapted species are tolerant by excluding and not by hyperaccumulating heavy metals. Indeed, heavy metals are only one of the several constraints occurring in those soils since plants also have to deal with a high Mg:Ca ratio, poor fertility and drought. Consequently, plants associated with such soils often have small and/or tough leaves [START_REF] Read | Sclerophylly in two contrasting tropical environments: low nutrients vs. low rainfall[END_REF]). The multiple independent origins of characters such as adaptation to ultramafic soils and heavy-metal hyperaccumulation within one given clade is paralleled by the multiple evolution of symbiosis allowing atmospheric nitrogen fixation in the 'nitrogenfixing clade' within Rosids [START_REF] Soltis | Chloroplast gene sequence data suggest a single origin of the predisposition for symbiotic nitrogen-fixation in angiosperms[END_REF], mycoheterotropy in Dioscoreales [START_REF] Merckx | Phylogeny and evolution of Burmanniaceae (Dioscoreales) based on nuclear and mitochondrial data[END_REF], C4 metabolisms in grasses [START_REF] Christin | C-4 photosynthesis evolved in grasses via parallel adaptive genetic changes[END_REF]

Exaptation to ultramafic soils

It might be hypothesized that the most represented clades in New Caledonia are those that present a potential pre-adaptation or rather an exaptation to ultramafic soils. According to [START_REF] Gould | Exaptation-a missing term in the science of form[END_REF], exaptations are "features that now enhance fitness but were not built by natural selection for their current role." The distinction between these two hypotheses lies in whether or not the plants that colonized New Caledonia were already growing on ultramafic soils on their original landmasses. Ultramafic soils are also found in Eastern Australia [START_REF] Batianoff | The serpentine flora of the humid subtropics of Eastern Australia[END_REF][START_REF] Batianoff | Vascular plant census and floristic analysis of serpentine landscapes in central Queensland[END_REF], but they cover a small inland surface and have a relatively small flora (552 indigenous species), that is not well differentiated. Only 18 species are restricted to the serpentine of Central Queensland, and only two nickel-hyperaccumulating species are recorded there [START_REF] Batianoff | Vascular plant census and floristic analysis of serpentine landscapes in central Queensland[END_REF]. In these areas, there is no record of several well diversified and over-represented families in New Caledonia, such as Cunoniaceae or Elaeocarpaceae, or the set of genera represented in the two areas is rather distinct for families such as Rubiaceae and Loganiaceae. However, it is possible that some groups used to be present on the Australian ultramafic soils and are not found there anymore. Furthermore, such soils are also recorded in areas such as the Philippines and New Guinea, but these floras are much less well studied [START_REF] Proctor | Vegetation and soil and plant chemistry on ultramafic rocks in the tropical Far East[END_REF] and less similar to that of New Caledonia [START_REF] Marsch | Large-scale patterns of plant diversity and conservation priorities in South-East Asia[END_REF]. Overall, most of these areas have one or several of the following features that suggest that they were not likely sources of propagules to colonize New Caledonia: small surface, poor flora, inland location, remoteness from New Caledonia. To our knowledge, no phylogenetic study to date has described a close relationship between any New Caledonian group and non-New Caledonian groups that grow on ultramafic soils. As indicated by the evolution of nickel-hyper-accumulation in distant areas in the genera Phyllanthus and Psychotria, cases of convergence are likely to be common. Thus, we believe that most plant groups were naïve to ultramafic soils when they reached New Caledonia, and their phylogenetic clustering would be evidence for exaptation of larger clades. Such groups would have been much more successful in colonizing New Caledonia and would have settled and diversified first. As erosion removed the layer of ultramafic soils, these groups that had already settled on the island would have had an advantage over long-distance, over-water dispersers in the invasion of adjacent non-ultramafic soils. Further phylogenetic studies are needed to see how many times each clade has colonized New Caledonia to tease apart the impact of ultramafic soils on their success in settling and their success in diversifying in New Caledonia.

Only a few studies suggest a role for some ecological filter for shaping island biota, apart from high dispersability. However, while studying plants of the California chaparral, [START_REF] Ackerly | Adaptation, niche conservatism and convergence: comparative studies of leaf evolution in the California chaparral[END_REF] found that small leaves, a generally accepted adaptation to Mediterraneantype climate, were already present in the ancestor of the chaparral lineage growing in cool temperate or subtropical conditions. He thus stated that "ancestors of chaparral taxa had already acquired appropriate traits that contributed to their success under Mediterranean-type climates." In the case of ultramafic soils, it has been suggested that plants adapted to drought may have a greater ability to grow on such soils [START_REF] Brady | Evolutionary ecology of plant adaptation to serpentine soils[END_REF]. Some similarities have been found in the response of plants to water stress and heavy metal stress, which can both involve small organic acids, amino acids and small peptides [START_REF] Bhatia | A role for nickel in osmotic adjustment in drought-stressed plants of the nickel hyperaccumulator Stackhousia tryonii Bailey[END_REF][START_REF] Sharma | The significance of amino-acids and amino-acid derived from molecules in plant responses and adaptation to heavy metal stress[END_REF][START_REF] Yang | OsMT1a, a type 1 metallothionein plays the pivotal role in zinc homeostasis and drought tolerance in rice[END_REF], and the exaptation to ultramafic soils may lie in these physiological pathways. Australian Cunoniaceae (36 species) and New Caledonian Asteraceae (34 species). Each bar represents the species richness of a genus, arranged in decreasing order. The seven genera of Cunoniaceae of New Caledonia probably represent each a single independent event of dispersal on New Caledonia [START_REF] Bradford | Phylogenetics and classification of Cunoniaceae (Oxalidales) using chloroplast DNA sequences and morphology[END_REF][START_REF] Bradford | Molecular phylogenetics and morphological evolution in Cunonieae (Cunoniaceae)[END_REF] with the exception of Spiraeanthemum, which likely arrived twice (Pillon et al. 2009b). Most of the Australian genera of Cunoniacae represent phylogenetic independent lineages [START_REF] Bradford | Phylogenetics and classification of Cunoniaceae (Oxalidales) using chloroplast DNA sequences and morphology[END_REF], and none of the New Caledonia genera of Asteraceae are endemic, representing at least as many events of colonization of New Caledonia. Therefore, diversification of New Caledonian Cunoniaceae was more intense than that of Australian Cunoniaceae and of New Caledonian Asteraceae. Asteraceae in Australia are represented by 899 species and 143 genera; the mean species number per genus is six and the largest genus has 99 species. Species numbers are taken from [START_REF] Hnatiuk | Census of Australian vascular plants[END_REF], [START_REF] Jaffré | Composition and characterisation of the native flora of New Caledonia[END_REF], [START_REF] Bradford | Cunoniaceae. The families and genera of vascular plants[END_REF] and [START_REF] Pillon | Biodiversité, origine et évolution des Cunoniaceae : implications pour la conservation de la flore de Nouvelle-Calédonie[END_REF].

  , and they indicate the existence of a phylogenetic signal in the composition of the New Caledonian flora. Phylogenetic independent contrasts showed a significant correlation between the hyperaccumulation index and the representation indexes for both indigenous (r=0.112, p=0.0094, d.f.=333) and endemic (r=0.199, p<0.001, d.f.=333) species. Thus, representation in the flora of New Caledonia was positively correlated with the occurrence of nickelhyperaccumulating species in families. The proportion of species endemic to New Caledonia within families was positively correlated with the representation index for indigenous (r=0.512, p<0.001) and endemic (r=0.738, p<0.001) species. The number of non-endemic species was negatively correlated with the representation index for indigenous (r=-0.118, p=0.07) and endemic (r=-0.176, p=0.015) species. DISCUSSION PHYLOGENETIC SIGNAL IN THE DISHARMONY OF THE NEW CALEDONIAN FLORA AND EVIDENCE FOR SPECIES SORTING The comparative analysis shows that the most represented and the least represented groups in the New Caledonian flora are not randomly distributed within the angiosperm phylogenetic tree. Some groups are particularly well represented and form the major component of the New Caledonian flora: the COM clade (Celastrales, Oxalidales and Malpighiales), Sapindales, Gentianales, Apiales, Ericales and Laurales. K values for representation indices for indigenous and endemic species and the occurrence of nickelhyperaccumulating species confirm the existence of a phylogenetic signal (Blomberg et al.

  , aerial parasitism in Santalales (Vidal-Russel & Nickrent 2008), among other examples.

Fig. 1 .

 1 Fig. 1. Contrasting pattern of diversification of New Caledonian Cunoniaceae (88 species),

Fig. 2 .

 2 Fig. 2. Representation of New Caledonian families mapped on the phylogenetic tree of the

Table 1 .

 1 Divergences making the largest contributions to the overall variation of the representation index. APA clade = Alseuosmiaceae+Phellinaceae+Argophyllaceae. COM clade = Celastrales+Oxalidales+Malpighiales. Nitrogen-fixing clade=

	Rosales+Fagales+Cucurbitales+Fabales
	Rank Divergences
		Indigenous species
	1	Magnoliids vs. Eudicots
	2	APA clade vs. Asteraceae+Calyceraceae+Goodeniaceae
	3	COM clade vs. nitrogen-fixing clade
	4	Paracryphiales vs. Asterales
		Endemic species
	1	Magnoliids vs. Eudicots
	2	APA clade vs. Asteraceae+Calyceraceae+Goodeniaceae
	3	Magnoliids+Eudicots vs. Monocots+Ceratophyllales
	4	COM clade vs. nitrogen-fixing clade

ACKNOWLEDGEMENTS

The authors wish to thank Nadia Robert, Isabelle Olivieri, Delphine Aigoin, and Elizabeth Stacy for reviewing earlier versions of the manuscript, Angela Moles for insights on seed size, and Hervé Jourdan for drawing attention to some references. Two anonymous reviewers, the handling editor, and the managing editor provided useful comments to improve the manuscript. This study was partly supported by the Agence Nationale de la Recherche (ULTRABIO, reference ANR-07-BDIV-010).

SUPPORTING INFORMATION

Additional supporting information may be found in the online version of this article: Appendix S1 List of families under-or over-represented in the New Caledonian flora.

Appendix S2

Representation of New Caledonian families mapped on the phylogenetic tree of the angiosperms, calculated for endemic species, or in comparison to the world flora.