Yohan Pillon

Helen C F Hopkins

Jérôme Munzinger

Hamid Amir

Mark W Chase

Cryptic species, gene recombination and hybridization in the genus Spiraeanthemum (Cunoniaceae) from New Caledonia

Keywords: Biodiversity hotspots, conservation of island endemics, hybridisation, island biogeography, single-nuclear copy gene, Pacific islands, serpentine, taxonomic inflation

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Cryptic species are defined as "two or more distinct species that are erroneously classified (and hidden) under one species name" [START_REF] Bickford | Cryptic species as a window on diversity and conservation[END_REF]. The importance of cryptic species probably varies among ecosystems, geographical areas and taxonomic groups, and few studies are available in the tropics. A few cases are known in higher plants [START_REF] Bickford | Cryptic species as a window on diversity and conservation[END_REF], including the Caesalpinia hintonii complex (Fabaceae, [START_REF] Sotuyo | Cryptic speciation in the Caesalpinia hintonii complex (Leguminosae: Caesalpinioideae) in a seasonally dry Mexican forest[END_REF] in Mexican dry forests, which contains two sibling species (sensu [START_REF] Bickford | Cryptic species as a window on diversity and conservation[END_REF] [START_REF] Guruschidze | Phylogenetic analysis of Allium subg. Melanocrommyum infers cryptic species and demands a new sectional classification[END_REF] and Cedrela (Meliaceae, [START_REF] Muellner | Molecular phylogenetics of Neotropical Cedreleae (mahogany family, Meliaceae) based on nuclear and plastid DNA sequences reveal multiple origins of "Cedrela odorata[END_REF] in which sibling species and cryptic species that are sometimes only distantly related have been identified in molecular phylogenetic analyses. Further occurrences have also been reported in Potamogeton (Potamogetonaceae, [START_REF] Whittall | Cryptic species in an endangered pondweed community (Potamogeton, Potamogetonaceae) revealed by AFLP markers[END_REF], Veronica (Plantaginaceae, [START_REF] Martínez-Ortega | Species boundaries and phylogeographic patterns in cryptic taxa inferred from AFLP markers: Veronica subgen. Pentasepalae (Scrophulariaceae) in the western Mediterranean[END_REF] and possibly Liparis (Orchidaceae, [START_REF] Pillon | Genetic diversity and ecological differentiation in the endangered fen orchid (Liparis loeselii)[END_REF], although in these cases the "cryptic species" revealed by molecular data were already described taxa and so were not really hidden, but rather poorly characterized. New examples of cryptic species are revealed every year with the use of molecular tools in a wide range of organisms [START_REF] Rieseberg | Editorial and retrospective 2008[END_REF], especially animals, but reports in flowering plants are still few. This is probably linked to the low variability observed in most markers used for molecular phylogenetics, which has also made DNA barcoding more difficult in plants than in animals [START_REF] Chase | A proposal for a standardised protocol to barcode all land plants[END_REF]. Nevertheless, methodologies used in population genetics would be expected to have revealed more cases of cryptic species in plants if they were common.

New Caledonia is an archipelago in the southwestern Pacific well known for its rich, unique and endangered flora, hence its recognition as a biodiversity hotspot for conservation priorities [START_REF] Myers | Biodiversity hotspots for conservation priorities[END_REF]. It is often considered that the richness of this flora is linked to the diversity of edaphic conditions found on the main island [START_REF] Jaffré | The relationship between ecological diversity and floristic diversity in New Caledonia[END_REF]. About one-third of the main island is covered with ultramafic soils [START_REF] Pelletier | Geology of the New Caledonia region and its implications for the study of the New Caledonian biodiversity[END_REF], 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 water-holding 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]. These soils, including those that are derived from "true" serpentine, usually present a challenging environment for plant development, but they are nevertheless covered by a species-rich vegetation in New Caledonia, with a high level of endemism, exceeding 90% [START_REF] Jaffré | Composition and characterisation of the native flora of New Caledonia[END_REF]. Other soil types in New Caledonia are often referred to as non-ultramafic, and they develop from rocks that include limestone, basalt, shale, etc. This basic dichotomy between ultramafic and non-ultramafic soils is a simple way to define the ecology of plant species because the majority are found on one or the other type, even though this may imply a disjunct or fragmented distribution. For instance, [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] calculated that 1176 species of seed plants were restricted to ultramafic substrates (see also [START_REF] Morat | Our knowledge of the flora of New Caledonia: endemism and diversity in relation to vegetation types and substrates[END_REF].

Nevertheless, species naturally found on both types of soils are not uncommon. Within Cunoniaceae, a family that has radiated in New Caledonia, some species with broad soil tolerance are monomorphic, e.g. Geissois hirsuta Brongn. & Gris, whereas others are complex and present considerable taxonomic challenges, e.g. Pancheria ternata Brongn. & Gris and Geissois pruinosa Brongn. & Gris (Hopkins & Pillon, unpublished manuscript). While investigating the molecular systematics of the genus Spiraeanthemum, also in the Cunoniaceae, we wanted to assess the validity of the circumscription of some species with wide substrate preferences. This genus encompasses 18 species of trees and shrubs distributed from the Moluccas to Samoa, including New Guinea, Queensland, the Solomon Islands, Vanuatu, Fiji, and New Caledonia, the last having the largest number of species [START_REF] Hoogland | Studies in the Cunoniaceae. II. The genera Caldcluvia, Pullea, Acsmithia and Spiraeanthemum[END_REF][START_REF] Hoogland | Studies in the Cunoniaceae. IV. Further notes on New Caledonian Acsmithia[END_REF]Pillon et al., 2009a). The genus can be divided into three groups (Pillon et al., 2009a), two of which occur in New Caledonia. The first (the S. densiflorum group) has a single New Caledonian species, S. densiflorum Brongn. & Gris, which is characterised by hairy apical buds, inflorescences branched near the base and several ovules per carpel; other species in this group are found in Australia and New Guinea. The S. brongniartianum group, characterised by a single ovule per carpel and pedunculate inflorescences, comprises six species in New Caledonia plus one, S. vitiense A. Gray, in Fiji.

The third group is Spiraeanthemum sensu stricto (S. samoense group), which occurs on several Pacific islands but not New Caledonia; its species have hairy apical buds, opposite leaves, pedunculate inflorescences and two ovules per carpel.

Of the six species in the Spiraeanthemum brongniartianum group in New Caledonia (Fig. 1), S. meridionale (Hoogland) Pillon and S. pedunculatum Schltr. are restricted to ultramafic soils in the south; S. collinum (Hoogland) Pillon has a scattered distribution on a few ultramafic massifs in the centre; S. brongniartianum Schltr. is widespread on nonultramafic soils; and S. pubescens Pamp. is also widespread but occurs on both soil types. Finally, S. ellipticum Pamp. is known from two mountains in the north on non-ultramafic soils, whereas in the south it occurs on several ultramafic ones, and it is unknown in between.

The disjunction in the range and ecology of S. ellipticum raises doubts about its circumscription.

In this study, we aimed to assess the circumscription of species in Spiraeanthemum using molecular markers. We selected for this purpose two nuclear low-copy genes, ncpGS and gapC, which have already been used successfully in Cunoniaceae (Pillon et al., 2009a;Pillon et al., 2009b). Low-copy nuclear genes are generally more informative than the most commonly used markers such as plastid or nuclear ribosomal loci because of their higher variability, biparental inheritance and general absence of concerted evolution [START_REF] Mort | The continuing search: low-copy nuclear sequences for lower-level plant molecular phylogenetic studies[END_REF][START_REF] Small | Use of nuclear genes for phylogeny reconstruction in plants[END_REF]. Previous works have shown that plastid genes display low levels of variation within genera of Cunoniaceae (Bradford, 2002, Pillon, unpublished), including within the S. brongniartianum group (Pillon et al., 2009a) and that multiple paralogues of ITS are recovered in several genera (Bradford, 2002, Pillon, unpublished).

MATERIALS & METHODS

All currently accepted species in the genus Spiraeanthemum (see Pillon et al., 2009a) in New Caledonia were sampled. The main focus of this study was the S. brongniartianum group in which multiple accessions per species were used whenever possible. Accessions of S. densiflorum (S. densiflorum group, New Caledonia) and S. macgillivrayi Seem.

(Spiraeanthemum s.s., Vanuatu) were used as outgroups. A previous study showed that the S. brongniartianum group was well-supported and that S. macgillivrayi and S. densiflorum were its successive sister groups, a set of relationships that was strongly supported (Pillon et al., 2009a). No material of S. vitiense (Fiji), the only species of the S. brongniartianum group occurring outside New Caledonia, was available. Leaf material 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 with a CTAB protocol modified from [START_REF] Doyle | A rapid DNA isolation procedure for small quantities of fresh leaf tissue[END_REF] by [START_REF] Csiba | DNA extraction protocols[END_REF]; the list of accessions is given in the appendix.

A portion of chloroplast-expressed glutamine synthetase (ncpGS), a low-copy nuclear 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]. The amplification mix included 45L of 2.5mM MgCl 2 Reddy PCR Master Mix 1.1× (ABgene Ltd), 1L of 0.4% bovine serum albumin (BSA), 0.5L of each primer (100ng/L) and 3L of template DNAs of unknown concentrations. The following amplification program was used: 2 min at 94°C, 38 cycles of 1 min at 94°C, 1 min at 50°C, 1 min 30 s at 72°C and a final extension of 5 min at 72°C. A portion of the glyceraldehyde 3-phosphate dehydrogenase (GapC, also known as G3pdh), another low-copy nuclear gene was amplified using the primers GPDX7F and GPDX9R from [START_REF] Strand | Nuclear DNA-based markers for plant evolutionary biology[END_REF] with the same amplification mix as for ncpGS. The following program was used: 2 min at 94°C, 38 cycles of 1 min at 94°C, 1 min at 53°C, 1 min at 72°C and a final extension of 5 min at 72°C.

No chromosome counts are available for Spiraeanthemum, but diploid chromosome numbers in other genera of Cunoniaceae vary between 24 and 32 [START_REF] Hopkins | Cunoniaceae[END_REF]. In Populus trichocarpa Torr. & A. Gray (Salicaceae), the most closely related model group to Cunoniaceae for which the entire genome has been sequenced [START_REF] Tuskan | The genome of black cottonwood, Populus trichocarpa Torr. & Gray[END_REF], one homeologue of GapC is found in linkage group I and homeologs of ncpGS are found in linkage groups VIII and X. Thus, the two genes are probably not linked in Spiraeanthemum.

If the electropherorams of directly sequenced amplification products of ncpGS indicated the presence of more than one allele in an accession and if these alleles differed in length or in more than one base, the PCR product was cloned. An average of six clones was routinely sequenced. Further clones were sequenced if all the base diversity observed in the electropherograms from the direct sequencing was not recovered. Satisfactory results from direct sequencing of GapC were not obtained, and thus amplification products from all accessions were cloned and sequenced (up to six clones per accession). For both genes, a consensus sequence was built for each cluster of clones on the phylogenetic tree in order to remove substitutions produced during PCR; these were compared with electropherograms from the direct sequencing when readable. PCR-induced recombinant sequences were detected by eye (with the help of the phylogenetic tree) and excluded. Sequences were submitted to Genbank (accession numbers GQ332607 to GQ332639), and full details are provided in the appendix.

Alignment was carried out easily by eye in a PAUP matrix, and parsimony analysis were carried out with PAUP*4.01b10 [START_REF] Swofford | PAUP*: Phylogenetic analysis using parsimony (and other methods) 4.0 Beta[END_REF] with DELTRAN character state optimisation. For GapC, the most parsimonious trees were recovered using branch-and-bound searches, and node support was measured with 1000 replicates of bootstrap, also with branchand-bound searches. For ncpGS, we used heuristic searches due to the larger size of the matrix, with TBR branch-swapping and MULTREES option on; node support was measured with 100 replicates using the same settings. Maximum likelihood analyses were carried out using PHYML [START_REF] Guindon | A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood[END_REF][START_REF] Guindon | PHYML online─a web server for fast maximum likelihood-base phylogenetic inference[END_REF] with a HKY model. This model best fitted all datasets according to Modeltest analyses [START_REF] Posada | MODELTEST: testing the model of DNA substitution[END_REF], except for the GapC dataset with recombinant sequences excluded, for which the K81uf model had a slightly lower AIC. Recombination networks [START_REF] Huson | Computing recombination networks from binary sequences[END_REF] were reconstructed using Splitstree4 [START_REF] Huson | Application of phylogenetic networks in evolutionary studies[END_REF] with default settings. Outgroups were not included in the networks since their inclusion leads to graphical distortion because they are on a long branch and we wanted to emphasize the ingroup (i.e. the S. brongniartianum group).

To test for the occurrence of recombination in our datasets, we used GARD software (Kosakovsky [START_REF] Pond | GARD: a genetic algorithm for recombination detection[END_REF] with a HKY85 substitution model, assuming no rate variation among sites. The latter setting proves the most sensitive considering the relatively low level of polymorphism observed in our datasets. Recombinant sequences were then identified by examination of the alignments. Further putative recombinant sequences were identified by progressive exclusion of sequences from the dataset until no crossovers were observed in the recombination networks. We then ran the phylogenetic analysis again excluding all putative recombinant alleles.

RESULTS

Detailed information on the ncpGS and GapC datasets is given in table 1. No sequences of GapC were obtained for Spiraeanthemum meridionale, but otherwise both genes were successfully amplified and sequenced for at least one accession of each New Caledonian species of Spiraeanthemum. Direct sequencing revealed that the accession Pillon et al. 20 of S. ellipticum had at least two alleles, but only one was successfully recovered by cloning.

Fewer accessions were sequenced for GapC because direct sequencing was not possible, and cloning was necessary in each case. We detected six likely PCR-induced recombinant sequences out of 59 clones (10.3%) in the ncpGS datset, and four out of 31 (12.9%) in the GapC dataset.

With the GARD analyses, several cases of recombination were detected in both ncpGS and GapC datasets. Identification of recombinant sequences required a detailed examination of the alignment, and by eye we detected six recombinant sequences in the ncpGS dataset and one in the GapC dataset. After exclusion of these recombinant sequences, GARD analyses no longer detected recombination in the ncpGS dataset, although they still did for GapC . The recombination networks (see below) indicate two further recombinant sequences in the ncpGS dataset and one in GapC. After exclusion of these recombinant sequences from the GapC dataset, recombination was still detected, but examination of the alignment indicates that recombination is not likely and that this may be a false positive result. Table 2 provides the base composition of the different accessions at the parsimony-informative sites for ncpGS.

Phylogenetic analyses of GapC sequences produce the same topology with both maximum parsimony and maximum likelihood, so only the first is shown (figure 2). In this phylogenetic tree, two groups can be distinguished. Group A consists of the northern accessions of both Spiraeanthemum pubescens and S. ellipticum, plus one of two alleles of S. collinum (allele 1). Group B contains S. brongniartianum, the southern accession of S. ellipticum, and allele 2 of S. collinum, which may be a recombinant sequence. For S. pedunculatum, allele 2 also belonged to group B, but allele 1 showed evidence of recombination around position 430; the first part of the alignment (positions 1-429) clustered with group A and the second part (positions 430-910) with group B.

In the ncpGS analysis, a well-supported group A was recovered in both maximum parsimony and maximum likelihood analyses (figure 3), containing the two northern accessions of both Spiraeanthemum ellipticum and S. pubescens. Group B formed a polytomy with group 1 nested in it and with little internal resolution; it contained the two accessions of S. brongniartianum, the single accession of S. collinum, the two southern accessions of S. ellipticum, both accessions of S. meridionale, both of S. pedunculatum and the single accession of southern S. pubescens. Several nodes were not present in the strict consensus of the maximum parsimony analysis, and these nodes were supported in the maximum likelihood analysis. We tentatively recognised five subgroups, B1 to B5, excluding the putative recombinant sequences. B1 is recovered in both analyses as part of the sister group to group A. Groups B2 and B3 were each supported in the maximum likelihood analysis with 85 and 76 bootstrap percentages, respectively. Putative recombinant sequences either formed clusters with subgroups B1 to B5 or independent clades. When all putative recombinant sequences were excluded from the ncpGS analysis, a better resolved phylogenetic tree was obtained using maximum likelihood (figure 4), with higher bootstrap percentages supporting group A and of subgroups B2 and B3. Group B was recovered (85 bootstrap percentage) but on a short branch. The topology obtained with the maximum parsimony analysis was the same except that group B was not monophyletic; subgroup B5 was recovered as sister to group A, but with low bootstrap support (<50%).

Recombination networks based on ncpGS and GapC sequence data are shown in figure 5. Groups A and B are present in both cases. In the GapC analysis, two crossovers were detected involving groups A and B, whereas in the ncpGS analysis, all crossovers detected were within group B, and almost no structure was observed within the latter, apart from subgroups B2 and B3. When outgroups were included, the rooting was imprecise because of the recombination events within the ingroup, but the outgroup tended to attach between groups A and B.

DISCUSSION

RECOMBINATION

Recombination events seem to have been common in New Caledonian Spiraeanthemum, especially in ncpGS, but also in GapC, which was less well sampled.

Because of the frequency of the phenomenon, it is difficult to tease apart clearly recombinant from non-recombinant sequences. Similarly high numbers of recombinant sequences were detected in Nicotiana (Solanaceae; Kelly et al., submitted). Recombination networks also detected putative cases of recombination not found by visual inspection of the alignments. As many as eight cases of recombination for ncpGS are suggested here, two of which possibly involved two breakpoints each. Some recombination may be associated with PCR and revealed only because of cloning (these recombinations would be unlikely to be observed in direct sequencing because they would be a minority signal); however, most alleles were cloned more than once. Examination of the direct sequences of S. meridionale shows that all differences between the two alleles occur in the last 163 base pairs, so in this case at least recombination is not an artefact; if differences are evident in direct sequencing, they are probably present in roughly 50% of the PCR copies, which is too frequent for them to be the result of PCR alone. Furthermore, no evidence of recombination was found in a phylogenetic study of Codia (Cunoniaceae) using the same set of markers and carried out at the same time (Pillon et al., 2009b), although several species of putative hybrid origins displayed multiple alleles.

It is not clear why recombination in ncpGS is so common in Spiraeanthemum. Many more cases were observed for this gene than for the less well sampled GapC (only two instances), and furthermore ncpGS is longer, so recombination events are more likely to occur. With a comparable level of variability, ncpGS offers more variable sites, which makes recombination easier to detect. We are not aware of cases of recombination for either gene reported previously in the literature. A possible explanation for the high numbers of recombination events is frequent hybridisation during the history of the genus (see below).

A first consequence of recombination is that it affects reconstruction of phylogenetic trees by increasing homoplasy. Recombination is expected to reduce the accuracy of phylogenetic analyses [START_REF] Posada | The effect of recombination on the accuracy of phylogeny estimation[END_REF], and indeed a large number of equally parsimonious trees were recovered, making the consensus tree highly unresolved. The exclusion of the recombinant sequences gives a much better resolved phylogenetic tree with good to strong support for most nodes, although some questions remain (e.g. monophyly of group B).

Within group B, most alleles recovered in the npGS analysis could either be assigned to one of four subgroups (B2, B3, B4 or B5) or were clearly recombinants between two alleles of these subgroups, i.e. they had no unique substitutions. However, allele 1 of S. collinum was assigned to subgroup B1 and did not show any signs of recombination when examined by eye; the recombination network indicates that it may nevertheless be recombinant. Several other alleles shared unique substitutions with it and have been assumed to be recombinant between this B1 allele and one from another subgroup. Thus, we may not have found the genuine B1 allele (i.e. with no recombination), and possibly the original sequence has been lost due to recombination. In general, the frequent occurrence of recombination may be homogenising the allele pool through the loss of some alleles. Identifying all the recombinant sequences was not straightforward; GARD analysis and recombination networks were not entirely in agreement about recombination. [START_REF] Schierup | Consequences of recombination on traditional phylogenetic analysis[END_REF] predicted that phylogenetic trees built on datasets including recombinant sequences would be more star-like with long terminal branches. The exclusion of recombinant sequences from the ncpGS dataset did reduce the number of long branches and most terminals fell in one of the major allele groups (groups A, B2, B3, in figure 5). Denser sampling should break up long branches and may help to clarify whether some putative cases of recombination detected are false and if other cases have been undetected.

To date few phylogenetic studies of plants have reported recombination in low-copy nuclear genes, possibly because this issue is not systematically explored, though exceptions include Cinnamoyl CoA reductase in Eucalyptus (Myrtaceae, [START_REF] Poke | The impact of intragenic recombination on phylogenetic reconstruction at the sectional level in Eucalyptus when using a single copy nuclear gene (cinnamoyl CoA reductase)[END_REF], PHOT (phototropin) in Verbena (Verbenaceae, [START_REF] Yuan | Evolution and phylogenetic utility of the PHOT gene duplicates in the Verbena complex (Verbenaceae): dramatic intron size variation and footprint of ancestral recombination[END_REF], and ncpGS, alcohol dehydrogenase and LEAFY/FLORICAULA in Nicotiana (Solanaceae, Clarkson et al., submitted; Kelly et al. submitted). Recombination will have to be considered as a potential pitfall of using low-copy nuclear genes in future phylogenetic studies.

EVIDENCE FOR THE EXISTENCE OF CRYPTIC SPECIES

Despite recombination, both analyses suggest the existence of two groups of alleles within Spiraeanthemum, A and B. Their distinctiveness is supported by a clear phylogenetic distance between them in both the ncpGS and GapC analysis. Group A is recovered in both, but group B is only well supported in the GapC analysis. In the ncpGS analysis, the accessions of northern S. ellipticum and S. pubescens both belong to group A, and those of southern S. ellipticum and S. pubescens cluster in group B. For GapC, where fewer samples were available, the single northern accessions of S. ellipticum and S. pubescens both fall into clade A, whereas the single accession of southern S. ellipticum is found in clade B. Thus, southern and northern populations of S. ellipticum and of S. pubescens appear to be genetically distinct. This is particularly clear for S. ellipticum, for which distinct northern and southern populations were observed in both gene trees, and two accessions in each geographical areas had a congruent placement in the ncpGS tree.

In terms of morphology, Spiraeanthemum ellipticum and S. pubescens are clearly distinct from one another and from other members of the genus. Spiraeanthemum ellipticum has glabrous resinous apical buds and sessile obovate leaves, whereas S. pubescens has hairy apical buds and lanceolate leaves with a distinct petiole. Both species can be shrubs or trees in scrub or forest, but S. ellipticum is restricted to high elevation (mostly 900-1600m), whereas S. pubescens generally occurs at low to medium elevation (mostly 0-700m). No morphological differences that could distinguish the northern and southern populations of either of these species have been found, and the main difference is in their ecology: the southern populations of both are found on ultramafic soils, and the northern populations on non-ultramafic substrates. The lack of morphological differences that correlate with geography, ecology and the patterns observed in molecular markers suggests that these may be examples of cryptic species.

Southern and northern populations of Spiraeanthemum ellipticum cannot be considered as sibling species (sensu [START_REF] Bickford | Cryptic species as a window on diversity and conservation[END_REF] because they are not each other's closest relatives according to the phylogenetic analyses presented here (figures 2 & 3). The large differences between alleles recovered in morphologically identical populations require an explanation. It would be puzzling if the same morphologies had evolved in parallel in groups A and B (figure 6, top diagram), but this is not the only possible scenario nor is it, in our view, the most likely.

An alternative hypothesis is that alleles of both groups A and B were originally present in the pool that included the common ancestors of both Spiraeanthemum ellipticum and S. pubescens; parallel lineage sorting in northern and southern populations then fixed alleles of groups A and B as observed here (Fig. 6, middle diagram). This could have occurred through either genetic drift or possibly natural selection driven by differences in the ecological conditions prevailing on non-ultramafic versus ultramafic soils. However, the same pattern of lineage sorting between two a priori unlinked genes seems unlikely, since lineage sorting is stochastic, but it can not be excluded.

A third possibility is that either the southern or the northern population, or both, underwent hybridisation and introgression (Fig. 6, bottom diagram). In this scenario, one or both populations would have acquired alleles from another species growing in the vicinity, and these new alleles would have replaced the ancestral ones of this species. Thus transgression within group A and/or group B would have obscured the histories of both Spiraeanthemum ellipticum and S. pubescens. If it seems improbable that such a process occurred independently in both regions producing by chance in these patterns, this scenario may be over-simplified because the other southern species with B alleles (S. meridionale and S. pedunculatum) may have also been involved. There are indeed similarities between some specimens of southern S. pubescens and S. meridionale, suggesting that species boundaries in these taxa may be porous [START_REF] Lexer | The use of digital image-based morphometrics to study the phenotypic mosaic in taxa with porous genome[END_REF]. Whatever the process involved, congruent allelic differences at two presumably unlinked loci between northern and southern populations of S. ellipticum lead us to conclude that they are now distinct but cryptic species.

IMPLICATIONS FOR THE EVOLUTION OF THE NEW CALEDONIAN FLORA

Overall, ecology correlated better with our molecular results than does morphology. In the tree based on maximum likelihood (Fig. 4), group A includes only accessions from populations occurring in the north on non-ultramafic soils (S. ellipticum p.p., S. pubescens p.p.), and all accessions growing on ultramafic soils have alleles belonging to group B.

According to the ncpGS phylogenetic tree from which recombinant sequences were excluded, adaptation to ultramafic soils may have evolved only once in Spiraeanthemum, in the clade containing groups B2, B3, and B5. This corroborates our findings in Codia (Pillon et al., 2009b), which showed that adaptation to ultramafic soils may not be as homoplasious as suggested by de Kok ( 2002) in other genera.

The complex geology of New Caledonia strongly constrains distributions of plants.

Most species are either restricted to ultramafic or non-ultramafic soils, and thus their distributions are generally expected to match one region on the geological map. Species recorded from both soil types represent a small portion of the flora. In Cunoniaceae, ca. 57% of the species are restricted or almost so to ultramafic soils, 28% are restricted to nonultramafic soils, and only 13% occur on both (Pillon & Hopkins, unpublished). The reported occurrence in taxonomic revisions of species on both ultramafic and non-ultramafic substrates may in some cases be due to misidentification, inadequate label data, or poor species delimitation, although complex local ecological and geological conditions or genuine ecological plasticity is likely to be involved in some instances.

The occurrence of two species of Spiraeanthemum, S. ellipticum and S. pubescens, that are both ecologically and genetically heterogeneous suggests that ecology should be given greater weight when evaluating species boundaries. The New Caledonian flora has largely been monographed using a strictly morphological definition for species, especially in older treatments that preceded most of the ecological studies of this vegetation. The presence of cryptic entities that correlate with soil preferences within a morphologically coherent "species" suggests that fidelity to soils may be higher than thought in the flora of New Caledonia. In Codia, species that occur on both soil types all have a hybrid origin (Pillon et al., 2009b), and it is possible that such hybrid species were formed on multiple occasions.

Two of these species (C. montana J.R. Forst. & G. Forst. and C. albicans Vieill. ex Pamp.) are clearly heterogeneous, but their patterns of morphological variation cannot be readily explained. The same is probably also true for some ecologically variable species in Geissois, Pancheria and Weinmannia (Hopkins & Pillon, unpublished manuscript).

If morphological entities with broad ecological preferences are actually more than one species, then the total number of taxa in the New Caledonian flora may be currently underestimated. It would be valuable to investigate more thoroughly the species with broad ecological preferences because in some cases less obvious morphological variation may correlate with ecology. Such an approach has been applied to Planchonella (Sapotaceae) in combination with molecular work [START_REF] Swenson | Molecular phylogeny of Planchonella (Sapotaceae) and eight new species from New Caledonia[END_REF], and the number of species recognised has significantly increased since the previous revision by Aubréville (1967). The application of genetics to the New Caledonian flora is thus likely to lead to a new form of "taxonomic inflation" [START_REF] Isaac | Taxonomic inflation: its influence on macroecology and conservation[END_REF]. However, it is unclear what proportion of ecologically variable taxa contains multiple species and therefore what is the contribution of cryptic species to the total diversity of the New Caledonian flora.

WHAT IS THE TRUE SPIRAEANTHEMUM ELLIPTICUM?

No obvious morphological characters distinguish southern and northern populations in either Spiraeanthemum ellipticum or S. pubescens. We cannot rule out the possibility that some morphometric or other more detailed study will identify differences in the future, but in general Spiraeanthemum offers relatively few morphological characters that differ at species level compared with Cunonia or Pancheria, for example. The three collections available from northern populations of S. ellipticum are inadequate for morphometric analysis as none is in full flower, and access to the only two localities known in the north (Mont Panié and Mont Colnett) is difficult.

The type of Spiraeanthemum ellipticum is Vieillard 2643 (lectotype: G ex DC, dated 1865; isolectotypes: A, BM, G, GH, K, L, MEL, NY, P, and Z, fide [START_REF] Hoogland | Studies in the Cunoniaceae. II. The genera Caldcluvia, Pullea, Acsmithia and Spiraeanthemum[END_REF]. The label gives the collection locality as "Fond de la rivière de Hienguen" (far end of the Hienghène river, presumably meaning the headwaters), which is south of Mont Panié, and no elevation is given. The sheets we have seen at K and P have one to seven fragments comprising leaves, flowers and/or fruits.

However, some doubt exists about the locality and as to whether Vieillard 2643 represents a single collection. Problems with locality data and mixed collections are common for Vieillard's specimens; his localities were often imprecise, and each of his collection numbers refers to material that he regarded as belonging to the same species and not necessarily to one particular gathering. Nevertheless, Hoogland considered the fragments on different sheets of Vieillard 2643 were sufficiently similar for him to cite them all as part of the type.

We are doubful about Vieillard's locality because no recent collections of this species have been made from the upper Hienghène river and the maximum elevation of the mountains on either side of the head of the valley (ca. 800 m) is lower than those where the plant has been collected on Monts Panié and Colnett (1000-1200 m, based on Pillon 347 & 616,Schmid 4136). If the locality is incorrect, it must then be doubtful whether the type came from the northern population of Spiraeanthemum ellipticum because we have no firm evidence that Vieillard ever visited Mont Panié or Mont Colnett.

Thus we cannot be sure from the label data that Vieillard 2643 is in fact from the northern population of Spiraeanthemum ellipticum, and we did not attempt to determine its likely origin by DNA analysis. DNA extraction from this old specimen would be challenging, and successful amplification of a low-copy nuclear gene needed to distinguish between northern and southern populations seems unlikely.

Because no morphological characters currently distinguish southern and northern populations of Spiraeanthemum ellipticum and because of problems with the type, we cannot alter its delimitation at present. Nevertheless, for conservation purposes it should be kept in mind that the populations in the two areas represent the results of distinct evolutionary events. This taxon is restricted to high elevation and may thus be considered threatened because of climate change, since the area available for it to occupy will decrease as vegetation zones move up the mountains due to global warming [START_REF] Pounds | Biological response to climate change on a tropical mountain[END_REF][START_REF] Thomas | Extinction risk from climate change[END_REF][START_REF] Thomas | Range retractions and extinction in the face of climate warming[END_REF][START_REF] Kelly | Rapid shifts in plant distribution with recent climate change[END_REF][START_REF] Munzinger | A second species in the endemic New Caledonian genus Gastrolepis (Stemonuraceae) and its implications for the conservation status of high-altitude maquis vegetation: coherent application of the IUCN Red List criteria is urgently needed in New Caledonia[END_REF]; and the northern population, currently known from only two mountains, is particularly at risk. Our sampling of Spiraeanthemum pubescens was not adequate to draw firm conclusions about whether more than one taxon should be recognised. Because no clear geographical gap exists between northern and southern populations, further sampling is needed, especially in the central part of its distribution.

EVIDENCE FOR HYBRIDISATION

The frequency of recombination in Spiraeanthemum suggests that hybridisation has probably been important in the history of this genus. For both genes investigated, we found clearly distinct alleles (excluding recombinants), suggesting that a period of genetic isolation allowed them to develop and become fixed in some populations. Thus, we think that recombination events occurred during secondary contact between species/populations.

In the GapC analysis, the single accession of Spiraeanthemum collinum and the accession of S. pedunculatum from Bois du Sud each displayed alleles or portion of alleles that belong to both groups A and B, suggesting a hybridisation event between these two population groups. However, this is not reflected in the ncpGS analysis where there was neither evidence of recombination events between alleles of group A and B nor any accession exhibiting alleles of both groups. However, both accessions of S. collinum and of S. pedunculatum displayed a mixture of alleles of subgroup B1, B2 and B3 in the ncpGS analysis. The true (ancestral) B1 allele may not have been recovered during this study, but the phylogenetic analysis suggests that it may be closer to group A than to the rest of group B.

Thus, these two accessions also appear to have a combination of alleles from group B and something close to group A, which would be a congruent scenario for the two datasets.

Furthermore, this would imply that some species might have had a hybrid origin. Similarly, one accession of S. ellipticum (Montagne des Sources) and one of S. pedunculatum (Pic du Pin) each produced one allele of subgroup B2 and one of subgroup B3, and it is also possible that these two species had hybrid origins. If this were the case, Spiraeanthemum pedunculatum might therefore have had two separate origins.

Hybrid speciation has been suspected in several other genera of Cunoniaceae from New Caledonia [START_REF] Pillon | Two new species of Cunonia (Cunoniaceae) from New Caledonia[END_REF]Pillon et al., 2009b), but considering that recombination has seriously obscured the history of Spiraeanthemum and that uncertainty surrounds the allele subgroup B1, hybrid speciation should only be considered as a possibility for this genus. Chromosome counts would also be valuable since they could provide information on the occurrence of polyploids, which are often associated with hybrid speciation [START_REF] Mallet | Hybrid speciation[END_REF], particularly when parental taxa are distantly related [START_REF] Paun | Hybrid speciation in angiosperms: parental divergence drives ploidy[END_REF].

Figure 1 .

 1 Figure 1. Distribution of the six species of Spiraeanthemum belonging to the S.

Figure 2 .

 2 Figure 2. One of the two most parsimonious trees based on the phylogenetic analysis of GapC

Figure 3 .

 3 Figure 3. Phylogenetic trees of Spiraeanthemum based on ncpGS sequence data. Left: one of

Figure 4 .

 4 Figure 4. Phylogenetic tree of Spiraeanthemum using maximum likelihood based on ncpGS

Figure 5 .

 5 Figure 5. Recombination networks for Spiraeanthemum (S. brongniartianum group) based on

Figure 6 .

 6 Figure 6. Possible scenarios for the origin of cryptic species in Spiraeanthemum. "Other

  within C. oyamae S. Sotuyo & G. P. Lewis that are geographically distinct. Other recently reported examples belong to the genera Allium (Alliaceae,

Table 1 .

 1 Information on the datasets for the two genes (ncpGS and GapC) included in the study.

		ncpGS	GapC
	Number of individuals	17	8
	Number of haplotypes	24	12
	Length of alignment	1209	906
	Variable sites	108 (8.9 %)	84 (9.3 %)
	Parsimony informative sites	64 (5.3 %)	52 (5.7%)

ACKNOWLEDGMENTS

The authors wish to thank the Direction de l'environnement (DENV, Province Sud) and Direction du développement économique et de l'environnement (DDEE, Province Nord) for allowing scientific collections in New Caledonia. We thank staff and students of the Botanical Laboratory at IRD for assistance in the field. Daniel and Irène Létocart, the late Henri Blaffart and the guides of the Dayu-Biik association, Steve Farino and Maurice Wanguene, also provided important assistance in the field and the herbarium. We thank the organisers of the Santo 2006 expedition in Vanuatu (IRD/MNHN/PRO-NATURA) during which material of Spiraeanthemum macgillivrayi was collected. Jason Bradford kindly provided some leaf material. We are grateful to Edith Kapinos, Laszlo Csiba, Olivier Maurin, Dion Devey, and Martyn Powell for their help in the Jodrell Laboratory and to Laura Kelly, Nina Rønsted, and Jim Clarkson for discussions about low-copy nuclear genes. Anaïs Cardineau and Cyril Poullain (CNRS) provided access to a Macintosh to carry out data analyses. Two anonymous reviewers made valuable comments on an earlier draft of the manuscript.

pubescens [N1] allele 1 T T A C T G T G G C C T T A G G T T G T A T A C A S. pubescens [N2] allele 2 T T A C T G T G G C C T T A G G T T G T A T A C A S. collinum allele 1 T T A T T T T G A C C T C A G A A T A C A T A G B1* S. ellipticum [S2] allele 1 T A A C T T T A A T -T C G G G A G A C G T T G B2 S. pedunculatum [1] allele 2 T A A C T T T A A T -T C G G G A G A C G T T G B2 S. pedunculatum [2] allele 2 T A A C T T T A A T -T C G G G A G A C G T T G B2 S. pubescens [S] T A A C T T T A A T -T C G G G A G A C G T T G B2 S. ellipticum [S2] allele 2 T T A C T T G G A T T T C G G G A T A C G T T G B3 S. pedunculatum [2] allele 1 T T A C T T G G A T T T C G G G A T A C G T T G B3 S. brongniartianum [S] allele 1 T T A C T T T G A T C C C G G G A T A C

collinum allele 2 T T A C T T G A A T -T C G G G A T A C G T T G B3+B2+B3 S. ellipticum [S1] allele 1 T A A C T T T A A T -T C A G A A T A C A T A G B2+B1 S. meridionale [1] allele 1 T A G C C T T G A T C T C G G G A T A C A T A G B5+B1 S. meridionale [2] T T A T T T T G A C C T C G G G A T A C G T T G B1+B5 S. pedunculatum [1] allele 1 T A A C T T T G A C C T C A G A A T A C A T A G B2+B1


Yohan Pillon

Helen C F Hopkins

Frédéric Rigault

Tanguy Jaffré

Elizabeth A Stacy

Cryptic adaptive radiation in tropical forest trees in New Caledonia

Keywords: Adaptive radiation, aluminium hyperaccumulator, island, New Caledonia, nickel hyperaccumulator, rainforest trees, serpentine, ultramafic

 The causes of the species richness of tropical trees are poorly understood, in particular the roles of ecological factors such as soil composition. The nickel-hyperaccumulating tree genus Geissois (Cunoniaceae) from the South-West Pacific was chosen as model of diversification on different substrates.

 We investigated the leaf element compositions, spatial distributions, and phylogeny of all species of Geissois occurring on New Caledonia.

 We found that New Caledonian Geissois descended from a single colonist and diversified relatively quickly into 13 species. Species on ultramafic and non-ultramafic substrates showed contrasting patterns of leaf element composition and range overlap. Those on non-ultramafic substrates were largely sympatric but had distinct leaf element compositions. In contrast, species on ultramafic substrates showed similar leaf element composition, but occurred in many cases exclusively in allopatry. Further, earlier work showed that at least three out of these seven species use different molecules to bind nickel.

 Geissois qualifies as a cryptic adaptive radiation, and may be the first such example in a lineage of tropical forest trees. Variation in biochemical strategies for coping with both typical and adverse soil conditions may help to explain the diversification and coexistence of tropical forest trees on similar soil types.

Introduction

The great diversity of species in tropical rainforests is a widely known fact and enigma, especially the coexistence, side by side, of many species of trees that have the same basic requirements. The coexistence of morphologically similar congeneric species in particular is puzzling [START_REF] Richards | Speciation in the tropical rain forest and the concept of the niche[END_REF][START_REF] Leigh | Why do some tropical forests have so many species of trees?[END_REF]. A growing number of studies indicate that substrates have a strong impact on the composition of tree communities and on the spatial distribution of individual plants within communities [START_REF] John | Soil nutrients influence spatial distributions of tropical tree species[END_REF][START_REF] Anderson | Soil-based habitat partitioning in understorey palms in lower montane tropical forests[END_REF][START_REF] Schreeg | Phylogenetic analysis of local-scale tree soil associations in a lowland moist tropical forest[END_REF][START_REF] Katabuchi | Soil resource availability shapes community trait structure in a species-rich dipterocarp forest[END_REF]. Further, soils have played a pivotal role in plant speciation in other vegetation types such as the fynbos and the succulent karoo of the Cape region [START_REF] Schnitzler | Causes of plant diversification in the Cape biodiversity hotspot of South Africa[END_REF]. Because most studies of rainforest plants have focused on communities, few have addressed niche partitioning among congeneric species. [START_REF] Fine | The contribution of edaphic heterogeneity to the evolution and diversity of Burseraceae trees in the western Amazon[END_REF] suggested that soils played a major role in the diversification of Protium in Amazonian forests; however, for sister species pairs that occur over large geographic scales (i.e. continents), it is difficult to exclude the possibility of earlier phases of allopatry in their divergence history.

In contrast, when diversification has occurred on small oceanic islands, ecological speciation with gene flow is the most logical explanation [START_REF] Savolainen | Sympatric speciation in palms on an oceanic island[END_REF][START_REF] Papadopulos | Speciation with gene flow on Lord Howe Island[END_REF], demonstrating the value of such systems to test this hypothesis.

New Caledonia, an archipelago in the south-west Pacific is renowned for its rich, unique and threatened flora [START_REF] Morat | The taxonomic reference base Florical and characteristics of the native vascular flora of New Caledonia[END_REF]. Although it is geologically a continental island, having drifted from Australia, New Caledonia was entirely submerged from the end of the Cretaceous until 37 My ago [START_REF] Cluzel | Upper Eocene unconformity and pre-obduction events in 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], and all studies to date support the postimmersion origin of its biota [START_REF] Grandcolas | New Caledonia: a very old Darwinian island?[END_REF][START_REF] Cruaud | Testing the emergence of New Caledonia: fig wasp mutualism as a case study and a review of evidence[END_REF][START_REF] Pillon | Time and tempo of diversification in the flora of New Caledonia[END_REF]. Thus, the history of the island's flora is comparable to that of true oceanic islands (e.g. volcanic islands) or "Darwinian" islands [START_REF] Grandcolas | New Caledonia: a very old Darwinian island?[END_REF] sensu [START_REF] Gillespie | Arthropods on islands: colonization, speciation, and conservation[END_REF].

Nonetheless, the rich flora of New Caledonia is short of dramatic examples of adaptive radiation, particularly when compared to the Hawaiian or Macaronesian Islands (see examples in [START_REF] Givnish | Ecology of plant speciation[END_REF]. Few radiations display the diversity of growth forms typically associated with ecological divergence. One notable exception is the genus Oxera (Lamiaceae), which contains a variety of life-forms: monocaule shrubs, tangled shrubs and vines that range from slightly to very woody [START_REF] Mabberley | Labiatae. In: Morat P ed. Flore de la Nouvelle-Calédonie et dépendances[END_REF]. Further, there are no convincing examples of radiations involving divergence in floral morphology associated with different pollination syndromes aside from Oxera, some orchids, and genera engaged in specific plant-pollinator mutualisms such as Ficus [START_REF] Cruaud | Testing the emergence of New Caledonia: fig wasp mutualism as a case study and a review of evidence[END_REF] and Phyllanthus (including Glochidion, [START_REF] Kawakita | Evolution of obligate pollination mutualism in New Caledonian Phyllanthus (Euphorbiaceae)[END_REF]. This paucity may be linked to the under-representation of lineages with zygomorphic flowers such as monocots, Asterales, Fabales, and Lamiales in New Caledonia [START_REF] Pillon | Ultramafic soils and species sorting in the flora of New Caledonia[END_REF].

The primary distinguishing feature of New Caledonia's terrestrial environment is the abundance of ultramafic substrates that cover one third of the surface of the main island. Soils that develop on ultramafic rocks (including serpentine) have several characteristics that are limiting for plant growth, including low availability of N, P and K, a high Mg/Ca ratio, high concentrations of heavy metals (e.g. Co, Cr, Ni) and variable but often low water-holding capacity; in addition, fire seems to play an important role in vegetation dynamics [START_REF] Jaffré | Etude écologique du peuplement végétal des sols dérivés de roches ultrabasiques en Nouvelle-Calédonie[END_REF][START_REF] Proctor | Vegetation and soil and plant chemistry on ultramafic rocks in the tropical Far East[END_REF]. Because of these edaphic characteristics, New Caledonia might be considered an Old Climatically Buffered Infertile Landscape (OCBIL, Hopper, 2009) or an Old Stable Landscape (OSL, [START_REF] Mucina | Landscape age and soil fertility, climate stability, and fire regime predictability: beyond the OCBIL framework[END_REF], where elevated nutritional specialization is expected. Ultramafic substrates have been implicated as a cause of the disharmonic nature of the New Caledonian flora, for example the under-representation of monocots and Lamiales or the overrepresentation of Magniolianae and Malpighiales [START_REF] Pillon | Ultramafic soils and species sorting in the flora of New Caledonia[END_REF]. Clades that are well represented in New Caledonia have a high frequency of nickel-hyperaccumulating species [START_REF] Jaffré | The metal hyperaccumulators from New Caledonia can broaden our understanding of nickel accumulation in plants[END_REF], a trait associated with ultramafic substrates [START_REF] Reeves | Metal-accumulating plants[END_REF]. The ability to grow on ultramafic substrates, and possibly metal hyperaccumulation, may have been exaptations [START_REF] Pillon | Ultramafic soils and species sorting in the flora of New Caledonia[END_REF]; i.e. these traits appeared in non-New Caledonian ancestors growing on normal soils and turned out to be advantageous when colonizing the island which was probably entirely covered with ultramafic rocks when it first emerged [START_REF] Pelletier | Geology of the New Caledonia region and its implications for the study of the New Caledonian biodiversity[END_REF].

Other types of substrate found in New Caledonia include limestone and mostly old metamorphic or sedimentary rocks (including greywacke, schist and shale) that can be hard to characterize. It is often accepted that the variety of New Caledonian soils is an important factor underlying the diversity of the flora [START_REF] Jaffré | The relationship between ecological diversity and floristic diversity in New Caledonia[END_REF]. How plants have adapted to these different edaphic conditions is poorly known, but the few studies that have addressed this problem suggest the involvement of high trait lability, hybrid speciation, and cryptic species among other possibilities [START_REF] De Kok | Are plant adaptations to growing on serpentine soil rare or common? A few case studies from New Caledonia[END_REF]Pillon et al., 2009b;Pillon et al., 2009c).

In this study, we focus on the genus Geissois (Cunoniaceae), a lineage of trees that are known to hyperaccumulate nickel [START_REF] Jaffré | Hyperaccumulation of nickel by Geissois species[END_REF]. We studied the leaf element composition and geographic distributions of 13 species in New Caledonia to examine associations among leaf element composition, soil type and degree of sympatry. Molecular phylogenetics and molecular clocks were also applied in order to test the single origin of the genus on the island, to estimate the speed of its diversification, and to determine if New Caledonian Geissois qualifies as an adaptive radiation.

Materials and Methods

The genus Geissois

The genus Geissois Labill. comprises 19 species, all occurring in Melanesia [START_REF] Hopkins | Karrabina gen. nov. (Cunoniaceae), for the Australian species previously placed in Geissois, and a synopsis of genera in the tribe Geissoieae[END_REF]: 13 species in New Caledonia, four in Fiji, one in Vanuatu, and one in the Temotu Province of the Solomon Islands (Fig. 1). Species of Geissois are tall shrubs to large trees primarily found in rainforests and forest edges but also in secondary vegetation, including savanna (generally dominated by Melaleuca quinquinervia) and maquis (scrubby vegetation on ultramafic rocks). The genus is characterized by its palmate, opposite leaves (3 to 11 leaflets), its conspicuous intrapetiolar stipules, and its relatively uniform flowers and inflorescences [START_REF] Hopkins | The morphology of stipules and inflorescences in Geissois sensu stricto (Cunoniaceae)[END_REF][START_REF] Fogliani | Morphological development of Geissois pruinosa (Cunoniaceae) from seed to adult, and the expression of plesiomorphic characters in seedlings[END_REF]. The flowers are red and show some minor variation in size and pubescence. They are arranged in bottle brush-like racemes that are axillary or borne on branches, and project stiffly from the stem or are sometimes pendulous. The racemes are simple (or trident in G. hirsuta), and may be solitary or in clusters. They attract a wide range of nectarivorous birds [START_REF] Barré | La faune des milieux sur roches ultramafiques[END_REF] as well as bats, geckos and insects (Hopkins et al., in press). The fruits are capsules, and the small winged seeds are dispersed by wind. Most species are distinguished by leaflet and stipule characters, including the number, size and shape of the leaflets, the size and shape of the stipules, and type of pubescence, as well as the presence of domatia (Hopkins et al., in press); none of these characters has any obvious adaptive value (cf [START_REF] Ashton | Speciation among tropical forest trees: some deductions in the light of recent evidence[END_REF].

Leaf element composition

To take into account recent advances in Geissois systematics [START_REF] Hopkins | Nomenclature and typification in Geissois (Cunoniaceae) in the South-West Pacific[END_REF][START_REF] Hopkins | Geissois bradfordii, a new species of Cunoniaceae from New Caledonia[END_REF][START_REF] Hopkins | Further new endemic taxa of Cunoniaceae from New Caledonia[END_REF], we updated the information in [START_REF] Jaffré | Hyperaccumulation of nickel by Geissois species[END_REF] with new measurements. Herbarium specimens have been shown to be a useful source of samples for the analysis of leaf element composition [START_REF] Brooks | Detection of nickeliferous rocks by analysis of herbarium specimens of indicator plants[END_REF]. Therefore, we obtained small leaf samples (~1g) at the Herbier de la Nouvelle-Calédonie (NOU) at IRD Nouméa. We sampled up to five specimens per species whenever possible, and more for species found on a range of soil types (Supporting Information Table S1). No samples were taken from herbarium sheets when doing so would have affected the quality of the information provided by that sheet, and this constrained our choice of samples for some species. Some species are naturally rare and represented by only a few specimens, e.g. the critically endangered G. belema and G. bradfordii, therefore fewer samples were included for such species. Otherwise, we tried to maximize the geographical coverage of each species' range. The samples were ground to a powder, and 0.25-0.3g was dry-ashed at 500°C for 5.5 hours and re-suspended in 25 mL of 1M HCl before being run on a Varian Vista MPX ICP-OES at the UH Hilo Analytical Lab. We measured concentrations of seven elements: Al, Ca, Cr, Fe, Mg, Ni, and P in each leaf sample. Samples with a value of iron above 500 ppm were conservatively excluded, because of possible soil contamination [START_REF] Reeves | Metal-accumulating plants[END_REF] recommended a threshold of 1000 ppm). Data were then log-transformed and standardized. We performed Non-Metric Multidimensional Scaling (NMDS) in PAST (Hammer et al., 2001) to obtain a graphical representation of the variation within and among species in leaf elemental content. We used Hotelling's pairwise comparison in PAST to test for differences in leaf element composition among species.

Species distributions

Species distributions were established from a list of specimens of Geissois (Hopkins et al., in press) largely based on the collection of the NOU herbarium. A convex polygon was drawn to represent the distribution of each species, and its surface area was calculated; overlap between those polygons and the ocean was nonexistent to negligible. We also calculated the percentage of geographic range overlap for each pair of species by dividing the area of overlap by the area of the species with the smaller range. Using the range of the more restricted species yielded the maximum estimate of sympatry, and thus the most conservative estimate of allopatry, in each case.

Phylogenetic analyses

For phylogenetic reconstruction, we supplemented and re-analyzed data from [START_REF] Pillon | Geissois (Cunoniaceae): another example of the Melanesian connection[END_REF]. Because of the persistence of multiple ITS copies in many Cunoniaceae, and the low level of variation of plastid genes, we sequenced a single plastid locus, trnL-F, and two nuclear genes, ncpGS and PHYC, using the protocols described in Pillon et al. (2009a). Sequences were available for all New Caledonia species known at that time plus Geisssois denhamii from Vanuatu. We added to this dataset trnL and PHYC sequences for the recently described G. belema and the Fijian G. ternata, and a trnL sequence of the Fijian G. superba from Genbank (Supporting information Table S4). Best fit substitution models for trnL, PHYC and ncpGS were HKY, HKY+ and HKY+ according to JModeltest analyses [START_REF] Posada | jModelTest: phylogenetic model averaging[END_REF]. No evidence for recombination was found in either the PHYC or the ncpGS datasets using GARD (Kosakovsky [START_REF] Pond | GARD: a genetic algorithm for recombination detection[END_REF], assuming no site-to-site variation (most sensitive setting). For each dataset, we ran two-million generations of Markov Chain Monte Carlo (MCMC) in MrBayes [START_REF] Ronquist | MrBayes 3: Bayesian phylogenetic inference under mixed models[END_REF], sampling the tree every 1,000 generations, using the appropriate substitution models and a 500,000-generation burnin; convergence was checked in Tracer (Rambaut & Drummond, 2007). In order to combine the PHYC and ncpGS datasets, where some species are represented by multiple accessions or heterozygotes, we used *BEAST [START_REF] Heled | Bayesian inference of species trees from multilocus data[END_REF]. We twice ran a 500-million-generation MCMC, with appropriate substitution models, a Yule process for tree reconstruction starting with a UPGMA tree, and a strict molecular clock, sampling one tree every 500,000 generations and with a burnin of 100-million generations.

In order to estimate a date for the origin of the radiation of New Caledonian Geissois, we applied a molecular clock analysis on a reduced combined dataset of ncpGS and PHYC. In spite of the availability of an almost complete generic sampling for plastid genes (Bradford & Barnes, 2001;[START_REF] Hopkins | Karrabina gen. nov. (Cunoniaceae), for the Australian species previously placed in Geissois, and a synopsis of genera in the tribe Geissoieae[END_REF] and the rich fossil record of Cunoniaceae in Australia [START_REF] Barnes | The history of Cunoniaceae in Australia from macrofossil evidence[END_REF], the absence of variation in plastid genes within New Caledonian Geissois (Supporting Information Fig. S4), as in other New Caledonian Cunoniaceae genera (Pillon, pers. obs.), precluded the dating of the crown radiation. To simplify the analyses, we limited the sampling within Geissois by avoiding heterozygote accessions but making sure to sample the deepest split within New Caledonian Geissois as well as two other genera of New Caledonian Cunoniaceae :

Codia and Spiraeanthemum (group brongniartianum, Pillon et al., 2009a). We ran two analyses of 100-million generations MCMC in BEAST v.1.7.5 [START_REF] Drummond | Bayesian phylogenetics with BEAUti and the BEAST 1.7[END_REF], applying appropriate substitution models, a lognormal clock, and a Yule tree prior using an input ultrametric tree obtained from r8s [START_REF] Sanderson | r8s: inferring absolute rates of molecular evolution and divergence times in the absence of a molecular clock[END_REF], sampling a tree every 100,000 generations and with a burnin of 10-million generations. Prior age at the root (divergence Cunoniaceae-Brunelliaceae) followed a normal distribution with a mean of 86 My and a standard deviation of 2 My. This age was obtained by [START_REF] Heibl | Distribution models and a dated phylogeny for Chilean Oxalis species reveal occupation of new habitats by different lineages, not rapid adaptive radiation[END_REF] for the divergence between Cunoniaceae and its putative sister group (Brunelliaceae, Cephalotaceae, Elaeocarpaceae). A similar age of 83 My was obtained independently for the same node by [START_REF] Xi | Phylogenomics and a posteriori data partitioning resolve the Cretaceous angiosperm radiation Malpighiales[END_REF]. All BEAST analyses were run on Lifeportal (University of Oslo), and convergence of the runs was checked in Tracer. Speciation rates were calculated using equation 4 of [START_REF] Magallón | Absolute diversification rates in angiosperm clades[END_REF].

Results

Low levels of iron were observed in all leaf samples of Geissois (max 230 ppm) with the exception of one sample of G. pruinosa var pruinosa (MacKee 45227, 924 ppm), which was excluded from subsequent analyses because of suspicion of contamination with soil. All species of Geissois naturally present on ultramafic substrates were found to be nickel hyperaccumulators (Ni content >1,000 ppm), with the notable exception of the recently described G. belema (Supporting Information Fig. S1). All species absent from ultramafic substrates had low nickel content, suggesting that the high levels reported previously in G. montana and G. racemosa by [START_REF] Jaffré | Hyperaccumulation of nickel by Geissois species[END_REF] were likely to result from confusion at a time where the taxonomy of the genus was not well understood. In fact, for G. hippocastanifolia, G. polyphylla and G. montana, nickel was undetectable in four out of five samples. Only one individual of G. balansae, from near Ponerihouen (Haudricourt 817, vague locality, no ecological information) had a relatively high nickel concentration (571 ppm). This finding suggests that this plant was growing on or in the vicinity of ultramafic substrate, although the species is not reported from this habitat. High nickel content was observed in all accessions of G. pruinosa var intermedia, a taxon that is reported only in the north-east, usually on non-ultramafic substrate. Lastly, we report here for the first time evidence that G. polyphylla is an Al hyperaccumulator; four of five individuals sampled had values equal to or above 1,000 ppm, and no other species of Geissois reached this threshold (see Apendix S5).

Non-Metric Multidimensional Scaling (NMDS) on all elements analyzed allowed a relatively clear separation between species found on ultramafic substrates and those found on non-ultramafic ones (Fig. 2). Overlaps were due to: G. hirsuta, which seems to be the only species unambiguously present on both soil types, the one sample of G. balansae with an unusually high Ni content, and G. belema, which grows on ultramafic substrates but does not hyperaccumulate nickel. Each of the five species restricted to non-ultramafic substrate had a largely distinct elemental profile, with only slight overlap between G. polyphylla and G. hippocastanifolia, and a larger overlap between G. montana and G. balansae. Although all of these species had similar nickel concentrations in their leaves, they varied considerably in the concentrations of other elements such as Al, Ca, Mg. Species from Vanuatu, Fiji and the Solomon Islands, all of which occur on non-ultramafic substrates, had elemental profiles that fell within the range of G. balansae (Supporting Information Fig. S2). The geographic ranges of species from non-ultramafic substrates in New Caledonia were highly overlapping (Fig. 3, Supporting Information Table S3 & Fig. S3). The only exception was the species pair, G. montana-G. balansae, the first of which occurred northward from Mt Aoupinié and the second southward from Mt Aoupinié. In contrast to the near-unique elemental profiles of non-ultramafic species, those restricted to ultramafic substrate showed highly overlapping leaf element compositions; no species had a distinct profile (Fig. 2), even if the widespread and ecologically variable G. pruinosa was excluded. Also in contrast to non-ultramafic species, the geographic distributions of taxa on ultramafic substrate tended to be more disjunct (Fig. 3, Supporting Information Table S3 & Fig. S3). For example, G. bradfordii and G. velutina are restricted to the far south, G. magnifica to the east coast, G. lanceolata to the west coast, and G. belema to the northern island of Art.

Little variation was found in the trnL dataset (Supporting Information Fig. S4) with all the New Caledonian species having identical DNA sequences except for G. hippocastanifolia, which differed by a single base; the two Fijian species shared one substitution. The PHYC tree (Supporting Information Fig. S5) was also poorly resolved with only two groups supported within Geissois: G. hippocastanifolia + G. polyphylla and G. denhamii (Vanuatu) + G. ternata (Fiji). In the ncpGS tree (Supporting Information Fig. S6), G. hippocastanifolia and G. polyphylla formed a clade that is sister to the rest of the species of New Caledonia plus G. denhamii (Vanuatu). Several species had multiple distant alleles, and some represented by multiple accessions were not monophyletic. The combined PHYC-npcGS analysis in *BEAST (Fig. 4) also recovered G. hippocastanifolia and G. polyphylla as sister species, forming a clade that was sister to the rest of the genus (G. racemosa group), including all other New Caledonian species and G. denhamii (Vanuatu). Internal supports within the G. racemosa group were low.

The molecular clock analysis on a combined PHYC-npcGS dataset (Supporting Information Fig. S7) indicated an age of 7.3 My (3.5-12.8 My) for the crown of Geissois (Table 1).

Discussion

New Caledonia is well known for its diverse flora; however, no dramatic cases of adaptive radiation have been documented thus far. We show here that Geissois satisfies the criteria of an adaptive radiation [START_REF] Schluter | The ecology of adaptive radiation[END_REF]: common ancestry, rapid speciation, correlation between phenotype and environment, and trait utility. The difference between New Caledonian

Geissois and classic examples of adaptive radiation is the lack of apparent niche separation in

Geissois, which occurs on just two broad soil types. Radiation of this genus within the small land area of New Caledonia's main island appears to have involved divergence of metal uptake mechanisms on ultramafic substrates and character displacement, or ultra-fine partitioning of edaphic niches, by species on non-ultramafic substrates.

Common ancestry-Molecular phylogenetic analyses provide support for the monophyly of Geissois; all New Caledonian species belong to a single clade and are likely to be descended from a single colonist. Subsequently, it seems that a unique dispersal event towards neighboring islands gave rise to the endemic species of Vanuatu and Fiji. Geissois polyphylla and G. hippocastanifolia are recovered as sister species, and are in turn sister to the Geissois racemosa group (all other New Caledonian species, including the morphologically divergent G. hirsuta and extra-New-Caledonian species). All species growing on ultramafic substrates belong to the G. racemosa group. This clade was poorly resolved, and several species were not recovered as monophyletic in the ncpGS analysis. The lack of cohesion of the individual species in this group suggests either hybridization among species or retention of ancestral polymorphisms, both of which are consistent with recent divergence.

Rapid speciation-Diversification of Geissois occurred at a rate of 0.26 sp.My -1 (Table 1). This rate is similar to that of other New Caledonian plant radiations [START_REF] Pillon | Time and tempo of diversification in the flora of New Caledonia[END_REF], which may be considered modest among known plant radiations. Nevertheless, when taking into account the small surface area of the island, Geissois had a diversification rate per unit area of 1.5 x 10 -5 sp.My -1 .km -2 . This is comparable to rates reported for rapid island radiations such as Hawaiian Bidens and Macaronesian Echium, and higher than the rates reported for large continental radiations such as Andean Lupinus and Eurasian Dianthus [START_REF] Knope | Area and the rapid radiation of Hawaiian Bidens (Asteraceae)[END_REF]. This figure is especially significant considering that the archipelago is dominated by one large island and is therefore far less fragmented and hence less suited for allopatric speciation than most archipelagos.

Correlation between phenotype and environment -Although the variation in vegetative traits that characterize the different species of Geissois in New Caledonia shows no clear relationship with ecology, the variation in leaf element composition among species is more readily associated with the environment. All Geissois species growing on ultramafic substrates except G. belema are nickel hyperaccumulators, species absent from non-ultramafic substrates have a higher Al content, and G. polyphylla qualifies as Al hyperaccumulator. As a group, species from ultramafic substrates were elementally distinct from those on non-ultramafic ones when considering all elements together. The contrast between these groups is not likely to be due solely to plasticity given that some species occurring sympatrically on ultramafic substrates, and thus likely experiencing equal nickel availability, displayed different levels of Ni concentration, for example G. velutina and G. pruinosa var pruinosa. In addition, previous greenhouse experiments found that the non-ultramafic G. montana is sensitive to watering with a nickel solution whereas the ultramafic taxon G. pruinosa var pruinosa is resistant (L. Richard, Y. Pillon & H. Amir pers. obs.), indicating a genetic basis for tolerance to this heavy metal. Although nickel tolerance and hyperaccumulation are different processes, the former is a pre-requisite for the latter, in natural settings at least. Understanding the mechanisms that underlie the variation among Geissois species in the ability to hyperaccumulate nickel will nevertheless require testing under controlled conditions.

Trait utility-Little is known about the evolutionary significance of nickel hyperaccumulation in Geissois. In other plant groups, however, nickel hyperaccumulation is suggested to confer resistance to herbivory [START_REF] Boyd | The defense hypothesis of elemental hyperaccumulation: status, challenge and new directions[END_REF], pathogens [START_REF] Hörger | The current status of the elemental defense hypothesis in relation to pathogens[END_REF] or drought [START_REF] Bhatia | A role for nickel in osmotic adjustment in drought-stressed plants of the nickel hyperaccumulator Stackhousia tryonii Bailey[END_REF]. There is less information on the significance of aluminium hyperaccumulation [START_REF] Jansen | Aluminium hyperaccumulation in Angiosperms: a review of its phylogenetic significance[END_REF], although it has been suggested as a mechanism to deter herbivory. This idea has not been well tested, however, and was not supported in a field study in the Philippines including six aluminium hyperaccumulator and one nickel hyperaccumulators [START_REF] Proctor | Foliar chemistry and leaf herbivory on Mount Bloomfield, Palawan, Philippines[END_REF]. In the aluminium hyperaccumulator, Melastoma malabathricum, an increase in oxalate concentration in the rhizosphere enabled solubilization of aluminium phosphate, and an increase in plant phosphorus was associated with an increase in plant aluminium [START_REF] Watanabe | Role of organic acids in aluminum accumulation and plant growth in Melastoma malabathricum[END_REF]. In the current study, the single aluminium hyperaccumulator species, G. polyphylla, also had the highest phosphorus content. It is possible, therefore, that this species has developed a similar mechanism to increase phosphorus uptake. Similarly nickel hyperaccumulation could also be a byproduct of a different physiological pathway rather than an end in itself.

Diversification on non-ultramafic substrates-Our data suggest character displacement within Geissois, at least on non-ultramafic substrates, where largely sympatric species exhibit different elemental signatures (Fig. 2, Supporting Information Table S5). The geology of New Caledonia is fairly complex, and non-ultramafic rocks of the main island include old and often poorly delimited geological layers. As such, we cannot exclude the possibility that different species of Geissois with overlapping distributions have preferences for different soils (micro-habitats), which could explain the variation in leaf element composition among them. However, in areas such as Col d'Amieu, G. balansae, G. polyphylla and G. racemosa can grow within a few meters of each other. Furthermore, G. hippocastanifolia and G. montana are often sympatric (Mandjélia, Aoupinié) and can hybridize as suggested by the intermediate morphology of one specimen (Pillon 81,Mandjélia). It is possible that these species differentially exploit available soil nutrients (cf. [START_REF] Richards | Speciation in the tropical rain forest and the concept of the niche[END_REF], and this may explain their coexistence. Testing this hypothesis would require analysis of individuals growing in controlled conditions. Interestingly the only two non-ultramafic species that have somewhat similar leaf element compositions, G. montana and G. balansae, are largely allopatric. Further, even though G. hirsuta has a broad ecology (occurring on both ultramafic and non-ultramafic substrates), its elemental signature is different from those of at least G. hippocastanifolia, G. montana and G. polyphylla (Fig. 2, Supporting Information Table S2). The fact that the allopatric G. denhamii (Vanuatu), G. ternata (Fiji) and G. pentaphylla (Solomon Islands) have relatively similar elemental signatures, that are in turn similar to the New Caledonian G. balansae, suggests that the variation in elemental signatures observed among species on non-ultramafic substrates in New Caledonia is not the result of random processes but rather of character displacement to minimize competition. The phylogenetic position of G. polyphylla indicates that aluminium hyperaccumulation is a derived character that evolved in New Caledonia, as it is not found in other species of the Geissois nor in the closely related Pseudoweinmannia and Lamanonia [START_REF] Nogueira | Foliar nutrient concentrations of tree species in four gallery forests in central Brazil[END_REF]. The character is, however, found in several less closely related genera of Cunoniaceae in Australia [START_REF] Jansen | Aluminium hyperaccumulation in Angiosperms: a review of its phylogenetic significance[END_REF].

Diversification on ultramafic substrates-Species growing on ultramafic substrates had largely overlapping elemental signatures, including high Ni content; however, the mechanisms by which they attain these signatures may be different. In a study of the nickel-binding ligand in several hyperaccumulating taxa, [START_REF] Callahan | Elemental and metabolite profiling of nickel hyperaccumulators from New Caledonia[END_REF] found that G. bradfordii, G. hirsuta and G. pruinosa var pruinosa had distinct metabolite profiles. Although in all species nickel was bound to nicotianamine, this metal was also bound to serine in G. bradfordii, to citric acid in G. pruinosa var pruinosa and to citric acid and galacturonic acid in G. hirsuta. Therefore, despite having similar levels of nickel in their tissues, these species appear to possess different mechanisms for nickel storage. Surprisingly, Geissois belema does not seem to accumulate nickel; this might be explained by the fact that it grows on highly weathered ferralitic soils where nickel is not available in significant amounts [START_REF] Jaffré | Conditions de milieu des terrains miniers[END_REF].

In contrast to non-ultramafic substrates that are almost continuous on the main island, ultramafic substrates represent a highly fragmented environment. Their patchy distribution across the island is likely to have favored allopatric speciation and may explain the weaker ecological differentiation observed in some cases, through reduced interspecific competition. Of all 13 species in New Caledonia, G. bradfordii is the only one that seems to have an obviously narrow ecology, being restricted to the bank of a river in an ultramafic area. This rupicolous species has narrow leaflets as expected in plants with such ecology [START_REF] Van Steenis | Rheophytes of the world[END_REF].

Interestingly, it occurs in the south of the island, as do some other rupicolous Cunoniaceae in the genera Spiraeanthemum, Cunonia and Pancheria (Hopkins et al., in press).

Widespread species-Only two species of Geissois are broadly distributed and documented on both ultramafic and non-ultramafic substrates. Geissois pruinosa is a species complex most commonly found on ultramafic substrates. Although several records suggested the presence of var intermedia on non-ultramafic rocks in the north-east, all of the samples analyzed in the current study had a high Ni content, suggesting that they were collected on ultramafic substrates.

Serpentine rocks are patchily distributed in the north-east, and it may be that G. pruinosa always has access to nickel and may be more strictly associated with ultramafic substrates than previously thought. Further fieldwork is required to re-evaluate the ecology of this species.

Geissois hirsuta is therefore the only species unambiguously present on both types of soil, and it is both morphologically distinct from other species and uniform throughout its range. Thus, the presence of cryptic species is not indicated (cf. Spiraeanthemum, Pillon et al., 2009b). Rather, it could represent an interesting model for studies of the genetic basis of adaptation to ultramafic conditions. Aside from these two widespread species, each species of Geissois possesses a unique combination of ecology, elemental signature, and geographic distribution (Table 2).

Therefore, only the mechanism by which these two species remain distinct remains unexplained by this study, although pollinators may play a role (see below).

Reproductive isolation-It is not clear how reproductive barriers evolved within Geissois to isolate so many species within such a small geographic area, but the variation observed in soils, leaf element composition and mechanism of metal accumulation may be associated with reduced fitness of interspecific hybrids (i.e., postzygotic barriers). Postzygotic barriers may then promote the evolution of prezygotic barriers through reinforcement [START_REF] Widmer | Evolution of reproductive isolation in plants[END_REF], which would be consistent with the differential flowering peaks among some Geissois species observed in the field but undetected in the herbarium (Hopkins et al., in press). Variation in flowering time may also be linked directly to differences in substrate [START_REF] Savolainen | Sympatric speciation in palms on an oceanic island[END_REF], particularly if associated with water availability [START_REF] Borchert | Soil and stem water storage determine phenology and distribution of tropical dry forest trees[END_REF]. With a single possible exception, there is no evidence of diversification in Geissois through adaptation to different pollinators. The uniquely widespread non-edaphic specialist G. hirsuta has distinct pendant, trident-shaped inflorescences that may attract a distinct suite of pollinators. Pollinator isolation might perhaps maintain the distinctiveness of this species in spite of its ecological and geographical overlap with so many other species of the genus. Further work is needed to test these ideas.

In conclusion, the radiation of Geissois on New Caledonia was the result of the rapid diversification from a single progenitor. Speciation was facilitated by the sharp contrast between non-ultramafic and ultramafic substrates where nickel-hyperaccumulating species evolved. On non-ultramafic substrates, species display broad geographic overlap but generally have distinct elemental signatures, suggesting specialization to different microhabitats within this variable soil type or, more likely, different preferences for soil nutrients. In contrast, on ultramafic substrates, allopatry may have been more important for diversification. However, even though species displayed similar elemental signatures, preliminary biochemical work suggests that different mechanisms evolved within Geissois to deal with ultramafic conditions. New Caledonia Geissois may be the first confirmed case of adaptive radiation in trees. There are only a modest number of confirmed cases of adaptive radiation in plants, and most involve herbs or plants that were ancestrally herbs [START_REF] Givnish | Ecology of plant speciation[END_REF]. Few studies have been able to determine how congeneric tree species partition the ecological space within a given geographical area [START_REF] Cavender-Bares | Multiple trait associations in relation to habitat differentiation among 17 Floridian oak species[END_REF]. Our study suggests that a large part of the diversity within a lineage of tropical forest trees may be hidden in their less-visible interactions with different soils and their different mechanisms of nutrient and metal uptake. These results need to be confirmed in other plant groups and in other geographical areas where contrasts in soil conditions may not be as sharp as in New Caledonia. , be= G. belema, br=G. bradfordii, hip=G. hippocastanifolia, hir=G. hirsuta, l=G. lanceolata, ma=G. magnifica, mo= G. montana, po=G. polyphylla, pr=G. pruinosa (all varieties), t= G. trifoliolata, ra= G. racemosa, v= G. velutina.

Figure 1 .

 1 Figure 1. Distributions of the genus Geissois in the South-West Pacific and the closely related

Figure 2 .

 2 Figure 2. NMDS of leaf element composition of New Caledonian Geissois species. ba =Geissois

Figure 3 .

 3 Figure 3. Example of distributions of selected Geissois species in New Caledonia, showing broad

Figure 4 .

 4 Figure 4. Half compatible consensus tree of Geissois obtained from the combined PHYC-ncpGS

Table 1 .

 1 Crown age for some Cunoniaceae radiations in New Caledonia. Diversification rate per km 2 is calculated for the main island only (16,372 km 2 ); Geissois belema, which is endemic to Art Island, was therefore excluded from the calculation of diversification rate.

		Species	Crown age median [95%	Diversific	Diversific
		number	confidence interval] (My)	ation rate	ation rate
				(sp.My -1 )	(sp.My -1
					km -2 )
	New Caledonian Geissois 13	7.3 [3.5-12.8]	0.26	1.5 10 -5
	Codia	14	5.3 [2.5-9.4]	0.37	2.2 10 -5
	Spiraeanthemum	6	6.3 [2.3-12.1]	0.17	1.1 10 -5
	group brongniartianum

Table 2 .

 2 Pairwise ecological, elemental and geographical differences between species of New Caledonia Geissois. S=no substrate overlap (ultramafic vs. non-ultramafic substrate), E=no elemental overlap (no overlaps between polygons in NMDS analysis of leaf element composition), e=minor elemental overlap (for the species pair G. hippocastanifolia/G. polyphylla), G=no geographical overlap of species distributions, g=geographical overlap of less than 5%. pruinosa includes both varieties (pruinosa, intermedia).

		balansae belema bradfordii hippocastanifolia hirsuta lanceolata magnifica montana polyphylla pruinosa racemosa trifoliolata velutina
	balansae
	belema	S/-/G
	bradfordii	S/E/G	-/E/G
	hippocastanifolia -/E/-	S/E/G	S/E/G
	hirsuta	-/-/-	-/-/G	-/E/-	-/E/-
	lanceolata	S/E/-	-/E/G	-/E/G	S/E/G	-/-/-
	magnifica	S/E/G	-/E/G	-/E/G	S/E/g	-/-/-	-/-/G
	montana	-/-/G	S/E/G	S/E/G	-/E/-	-/E/-	S/E/G	-/E/G
	polyphylla	-/E/-	S/E/G	S/E/G	-/e/-	-/E/-	S/E/g	S/E/G	-/E/-
	pruinosa	-/-/-	-/E/G	-/-/-	-/E/-	-/-/-	-/-/-	-/-/-	-/E/-	-/E/-
	racemosa	-/E/-	S/E/G	S/E/G	-/E/-	-/-/-	-/E/-	S/E/-	-/E/-	-/E/-	-/E/
	trifoliolata	-/E/-	-/E/G	-/E/G	-/E/G	-/-/-	-/E/G	-/-/G	-/E/G	-/E/-	-/-/-	-/E/-
	velutina	S/-/g	-/E/G	-/E/-	S/E/G	-/-/-	-/E/G	-/-/G	S/E/G	S/E/G	-/-/-	S/E/G	-/E/G

Acknowledgments

We wish to thank Laure Barrabé for her assistance at many stages of this work. We thank Jason Bradford, Eve Lucas, Jérôme Munzinger and the DNA banks at Royal Botanic Gardens, Kew and Missouri Botanical Garden for providing leaf material/DNA samples. Material from Vanuatu was collected during the joint IRD/MNHN/Pro-Natura expedition Santo 2006. We thank Jacqueline Fambart-Tinel and the staff of the herbarium at NOU for assistance in the field and for allowing sampling from specimens. We thank Tomoko Sakishima and Jodie Schulten for assistance in the lab, as well as Anne Veillet (Genetics Core Facility, UH Hilo) and Lucas Mead (Analytical Laboratory, UH Hilo) for processing sequencing and chemical analyses. Finally, we thank Becky Ostertag for helpful discussions and three anonymous reviewers for their comments on an earlier version of this manuscript. Funding for chemical analyses was provided by NSF DEB 0954274.

Supporting information

Table S1. Results of leaf element analyses on dry leaf samples. Table S2. Results of Hotelling pairwise comparisons of leaf element compositions of Geissois species.

Table S3. Pairwise percentage overlap of the geographic distributions of New Caledonian Geissois species. Table S4. Index of collections used for molecular phylogenetics analyses with voucher information and Genbank accession numbers. Table S5. Principal ecological and elemental characteristics of Geissois species mentioned in the text.


Geissois (Cunoniaceae), another example of the Melanesian connection

Yohan Pillon

The genus Geissois, taken in the strict sense (i.e., excluding the Australian species), a member of the family Cunoniaceae, is a group of trees and shrubs

  endemic to the islands of the Pacific. It is easily distinguished by its opposite palmately compound leaves and its bright red flowers arranged in bottle-brush like inflorescences (Figure 1

Figure 1 .

 1 Figure 1. Geissois denhamii above Penaorou camp, Santo

Figure 2 .

 2 Figure 2. Distribution of the species of Geissois within Melaneisa

Figure 3 .

 3 Figure 3. Phylogeny of the genus Geissois according to molecular data