Yohan Pillon

Jérôme Munzinger

Hamid Amir

Helen C F Hopkins

Mark W Chase

Keywords: New Caledonia, biogeography, hybrid speciation, island, rainforest refugia, serpentine

We reconstructed the evolutionary history of Codia, a plant genus endemic to the New Caledonia biodiversity hotspot in the south-west Pacific, using three single-copy nuclear genes. It seems likely that more than half of Codia species have a hybrid origin, but in the absence of cytological information, it is not known whether polyploids occur. Adaptation to ultramafic soils is possibly a plesiomorphic character for the entire genus. We found that species of hybrid origin can have some morphological characters absent in putative parental species, i.e. they exhibit transgressive phenotypes. There is evidence of considerable range alteration post-origin in several species because some likely parental species of hybrid taxa no longer co-occur and are confined to putative rainforest refugia; in some cases, hybrid species do not now co-occur with either of their parental species. These results have implications for the design of conservation strategies, for example prioritization of parental species for ex-situ conservation and preservation of the contact zones between soil types where hybridization is more likely to occur (i.e. conserving the possibility for the process to continue rather than trying to conserve taxa).

INTRODUCTION

Identification of the best strategies for conservation requires not only better knowledge of biodiversity but also better understanding of the evolutionary processes involved in its production [START_REF] Erwin | An evolutionary basis for conservation strategies[END_REF][START_REF] Smith | The preservation of process: the missing element of conservation program[END_REF][START_REF] Cowling | Rapid plant diversification: planning for an evolutionary future[END_REF][START_REF] Moritz | Strategies to protect biological diversity and the evolutionary processes that sustain it[END_REF].

Among factors involved in plant speciation, two may be considered particularly important: hybridization [START_REF] Rieseberg | Hybrid origins of plant species[END_REF] and polyploidization [START_REF] Ramsey | Pathways, mechanisms, and rates of polyploid formation in flowering plants[END_REF]. A large number of model groups have been the subject of studies of hybridization and/or polyploidization, but substantial biases exist in their choice. Many are crops, ornamentals or weeds, and most have one or more of the following characteristics: brief life history, easy cultivation, and temperate climatic preferences (with biases towards native plants of Europe and North America). Although most biodiversity lies in the tropics, comparatively few data are available for tropical plants, particularly woody taxa.

New Caledonia is an archipelago in the south-western Pacific well-known for its rich and unique flora [START_REF] Jaffré | Composition and characterisation of the native flora of New Caledonia Institut de Recherche pour le Développement[END_REF], and considered by Meyers et al. (2000) as a global biodiversity hotspot. Its endemic flora is composed of a large number of relict lineages, such as conifers, early diverging angiosperms (including Amborella), and magnoliids [START_REF] Jaffré | Composition and characterisation of the native flora of New Caledonia Institut de Recherche pour le Développement[END_REF], as well as major radiations in genera such as Phyllanthus (114 species), Psychotria (87 species) and Syzygium (68 species). A factor often used to explain the peculiarity of the New Caledonian flora is the importance of the ultramafic soils that cover roughly one-third of the main island's surface [START_REF] Jaffré | The relationship between ecological diversity and floristic diversity in New Caledonia[END_REF]. Ultramafic soils, including serpentine in the strict sense, 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.), low water-holding capacity and increased susceptibility to fire [START_REF] Proctor | Vegetation and soil and plant chemistry on ultramafic rocks in the tropical Far East[END_REF]. In New Caledonia, large areas of ultramafic substrates are covered by scrubby vegetation termed "maquis" in which levels of species endemism can exceed 90% [START_REF] Jaffré | Composition and characterisation of the native flora of New Caledonia Institut de Recherche pour le Développement[END_REF], especially at high elevation.

Relatively few studies have investigated diversification of particular groups within New Caledonia (but see [START_REF] Eibl | Evolution of Polyscias sect. Tieghemopanax (Araliaceae) based on nuclear and chloroplast DNA sequence data[END_REF][START_REF] De Kok | Are plant adaptations to growing on serpentine soil rare or common? A few case studies from New Caledonia[END_REF][START_REF] Swenson | Molecular phylogeny of Planchonella (Sapotaceae) and eight new species from New Caledonia[END_REF] compared with areas such as Hawaii [START_REF] Price | Speciation in Hawaiian angiosperms lineages: cause, consequence and mode[END_REF] or the Cape region of South Africa [START_REF] Linder | Evolution of diversity: the Cape flora[END_REF]. Molecular phylogenetics is an ideal tool to evaluate evolutionary processes within endemic groups, but the choice of adequate markers is an important issue. Plastid genes and nuclear ribosomal regions are not the most appropriate genomic regions for tracking hybridization [START_REF] Small | Use of nuclear genes for phylogeny reconstruction in plants[END_REF] because the first are generally inherited uniparentally (generally maternally in angiosperms, [START_REF] Corriveau | Rapid screening method to detect potential biparental inheritance of plastid DNA and results for over 200 angiosperm species[END_REF], and the second are subject to concerted evolution [START_REF] Baldwin | The ITS region of nuclear ribosomal DNA: a valuable source of evidence on angiosperm phylogeny[END_REF], which tends to erase one of the parental copies in species of hybrid origin [START_REF] Chase | Molecular systematics, GISH and the origin of hybrid taxa in Nicotiana (Solanaceae)[END_REF]. In many cases, concerted evolution in hybrids is strongly biased towards the maternal parent [START_REF] Pillon | Evolution and temporal diversification of western European polyploid species complexes in Dactylorhiza (Orchidaceae)[END_REF], and thus both these categories of markers may indicate congruent evolutionary histories leaving no evidence of hybridization. Single-copy nuclear genes provide better markers for this task, although use of such loci is often difficult due to the lack of universal primers and their often unpredictable modes of evolution.

To investigate processes that have been important in diversification of the New Caledonian flora, and particularly to examine the importance of soil diversity and the possible occurrence of hybridization, we aimed to reconstruct the evolutionary history of the endemic genus Codia. This genus belongs to Cunoniaceae, a principally Southern Hemisphere family of 27 genera and ca. 300 species [START_REF] Bradford | Cunoniaceae. In: The families and genera of vascular plants[END_REF]) that is well represented in New Caledonia. Codia consists of some 14 species [START_REF] Hopkins | Nomenclature and typification in the endemic genus Codia (Cunoniaceae) from New Caledonia[END_REF][START_REF] Hopkins | Four new species in the endemic genus Codia (Cunoniaceae) from New Caledonia[END_REF] of often gregarious trees and shrubs that sometimes dominate maquis and rainforests. With species on both ultramafic and non-ultramafic (e.g. sedimentary, basaltic and metamorphic) substrates, Codia comprises both widespread and micro-endemic species on the main island (Grande Terre), Yandé and the Belep islands. Species delimitation has been problematic; little variation is seen in reproductive morphology, but species are often variable in vegetative characters and in some cases hybridization has been suspected (e.g. [START_REF] Hopkins | Four new species in the endemic genus Codia (Cunoniaceae) from New Caledonia[END_REF].

Through reconstruction of the evolutionary history of this genus using molecular markers, we hoped to gain insights into its morphological and ecological diversification, its biogeography, and the processes that may have triggered this radiation, which would help to demonstrate how this process may be conserved.

MATERIAL & METHODS

All but one species currently recognized in Codia according to recent taxonomic works [START_REF] Hopkins | Nomenclature and typification in the endemic genus Codia (Cunoniaceae) from New Caledonia[END_REF][START_REF] Hopkins | Four new species in the endemic genus Codia (Cunoniaceae) from New Caledonia[END_REF] were sampled in this study. Material was unavailable for just C. cinerascens, known only from the type collection made in the 19 th century that indicates a vague locality ("Montagnes d'Ouatendé, Gatope"). In spite of extensive field work, no plant matching this type was found. Although the type of C. cinerascens is morphologically distinctive, the broad stipules and minute indumentum on the leaves recall C. albicans, and it may be an anomalous form of this species or a cross between it and perhaps C. spatulata (Hopkins & Pillon, unpublished). A single accession was used for each species. Because of technical limitations, we chose to multiply the markers rather than the accessions to increase the resolution, since preliminary studies showed that the level of molecular variation within Codia was low. Species of Spiraeanthemum and Geissois were used as outgroups [START_REF] Pillon | A molecular and morphological survey of generic limits of Acsmithia and Spiraeanthemum (Cunoniaceae)[END_REF]Pillon, upublished).

Preliminary studies showed that classic markers would be of limited use for the present study. For instance, multiple copies of ITS were recovered in most species of Cunoniaceae [START_REF] Bradford | Molecular phylogenetics and morphological evolution in Cunonieae (Cunoniaceae)[END_REF]Pillon, unpublished). For plastid genes, little variation was found within the genera of this family [START_REF] Bradford | Molecular phylogenetics and morphological evolution in Cunonieae (Cunoniaceae)[END_REF][START_REF] Pillon | A molecular and morphological survey of generic limits of Acsmithia and Spiraeanthemum (Cunoniaceae)[END_REF]Pillon, unpublished), and the level of variation in two candidate genes for plant DNA barcoding [START_REF] Kress | Use of DNA barcodes to identify flowering plants[END_REF][START_REF] Chase | A proposal for a standardised protocol to barcode all land plants[END_REF], matK and psbA-trnH, is also low (Pillon, unpublished). This led us to search for suitable single-copy genes. Ideally, the choice of such genes should be based on a number of criteria, including a high-level of variation and the absence of similar orthologous genes, duplication, and recombination [START_REF] Small | Use of nuclear genes for phylogeny reconstruction in plants[END_REF].

However, when working on non-model organisms, one constraint is the availability of primers that allow amplification of such genes. We thus selected genes that had previously been used in groups that are closely related to Cunoniaceae: ncpGS (chloroplast-expressed glutamine synthetase) was used in Oxalis (Oxalidaceae, [START_REF] Emshwiller | Chloroplast-expressed glutamine synthetase (ncpGS): potential utility for phylogenetic studies with an example from Oxalis (Oxalidaceae)[END_REF], which belongs to Oxalidales, the same order as Cunoniaceae; GapC (glyceraldehyde 3-phosphate dehydrogenase, also known as G3pdh) was used in Manihot (Euphorbiaceae, [START_REF] Olsen | Evidence on the origin of cassava: phylogeography of Manihot esculenta[END_REF]; and the first exon of PHYC (phytochrome C) was used in Phyllanthaceae [START_REF] Samuel | Molecular phylogenetics of Phyllanthaceae: evidence from plastid matK and nuclear PHYC sequences[END_REF]; the latter two families both belong to Malpighiales, an order close to Oxalidales [START_REF] Zhu | Mitochondrial matR sequences help to resolve deep phylogenetic relationships in rosids[END_REF]. In Populus trichocarpus, the most closely related model group for which the entire genome has been sequenced [START_REF] Tuskan | The genome of black cottonwood, Populus trichocarpa (Torr. & Gray)[END_REF], one homolog of GapC is found in linkage group I and homologs of ncpGS are found in linkage groups VIII and X. These two genes are thus not linked in Populus, and PHYC is missing in this model species [START_REF] Howe | Evidence that the phytochrome gene family in black cottonwood has one PHYA locus and two PHYB loci but lacks members of the PHYC/F and PHYE subfamilies[END_REF].

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 modified CTAB protocol [START_REF] Csiba | DNA and tissue banking for biodiversity and conservation: theory, practice and uses[END_REF]. A portion of ncpGS 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 protocol 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 PHYC was amplified using the primer PHYC-F and PHYC-R from [START_REF] Samuel | Molecular phylogenetics of Phyllanthaceae: evidence from plastid matK and nuclear PHYC sequences[END_REF]. The amplification mix included 45L of 1.5mM MgCl 2 Reddy PCR Master Mix 1.1× (ABgene Ltd), 1.5L of 0.4% bovine serum albumin (BSA), 0.5 L of each primer (100ng/L), 1 L of DMSO and 2L of template DNAs of unknown concentrations.

The following protocol 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. A portion of GapC was amplified using the primer GPDX7F and GPDX9R from [START_REF] Strand | Nuclear DNA-based markers for plant evolutionary biology[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 protocol 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.

In the case of ncpGS, the amplification product could generally be sequenced directly.

If electrophoregrams showed the presence of more than one allele/copy in an accession, and if these alleles differed in length or by more than one base pair, then the PCR product was cloned. An average of six clones were routinely sequenced. Further clones were sequenced if all polymorphisms observed in the electrophoregrams from the direct sequencing were not recovered. PHYC was also able to be sequenced directly. When more than one allele was observed at this locus, sequences of these alleles/copies were deduced by comparison with alleles found in the other accessions that were homozygous. This was necessary for only two accessions. When directly sequenced, GapC did not give clean results, and therefore amplification products of all accessions were cloned (up to six clones sequenced per accession in order to account for the heterogeneity observed). For this marker, a consensus sequence was created for each uniquely clustering set of clones, which removed the effects of substitutions induced by the PCR and cloning procedures. For ncpGS, the comparison between cloned and direct sequences allowed removal of most such artefacts. DNA sequences have been submitted to Genbank (accession numbers FJ595023-FJ595081, see supplementary data for full details).

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]. We searched for putative recombination events using the GARD software [START_REF] Pond | GARD: a genetic algorithm for recombination detection[END_REF]. A distance matrix between alleles was built with MEGA [START_REF] Kumar | MEGA 3: Integrated software for molecular evolutionary genetic analysis and sequence alignment[END_REF] and used to create a network with TCS [START_REF] Clement | TCS: a computer program to estimate gene genealogies[END_REF]. To represent relationships between the species of Codia using all three DNA regions ("total evidence" approach), distances between species were calculated using POFAD [START_REF] Joly | Incorporating allelic variation for reconstructing the evolutionary history of organisms from multiple genes: an example from Rosa in North America[END_REF] and used to build a split network [START_REF] Huson | Application of phylogenetic networks in evolutionary studies[END_REF].

RESULTS

The total length of the aligned ncpGS matrix was 1062 base pairs (bp); 54 sites were variable, 12 of which were potentially parsimony informative. The aligned matrices of PHYC and GapC were 579 and 873 bp long, respectively; they displayed 9 and 46 variable sites and provided 8 and 10 potentially parsimony informative sites, respectively. No evidence of recombination in any of the sequences was found with the GARD software.

Networks for alleles of ncpGS, PHYC and GapC found in Codia are shown in figure 1, and table 1 shows the distribution of these alleles among species. For ncpGS and GapC, up to four allele groups could be distinguished in the network, and these alleles were designated A, I, J and N, corresponding to the following species: C. albicans, C. incrassata, C. jaffrei and C. nitida. For PHYC, no obvious clear-cut groups were found, in contrast to the situation for the two other genes, and two groups (I and J) were tentatively delimitated by comparison with these other genes. For none of the loci did we ever recover more than two alleles in a given accession.

Four species had genomes with alleles belonging to single allele-groups. In Codia jaffrei, we recovered a single allele (group J) for all three genes; in C. incrassata, we also recovered a single allele (group I) for all three genes. For C. ferruginea, a single allele was recovered for ncpGS and PHYC, but two similar alleles (with a difference of two substitutions between them) were found for GapC; all these alleles belonged to group I. Codia nitida produced two similar but unique alleles (group N) for ncpGS; for PHYC, it had the J1 allele, and for GapC, the N1 allele, which is nested in group I. A phylogenetic tree based on the combined analysis of the three genes for these four species using maximum parsimony showed that C. nitida and C. jaffrei form a clade (73 bootstrap percentage, BP) that is sister to another clade composed of C. ferruginea and C. incrassata (99 BP, data not shown).

For each of the other nine species of Codia, we recovered in at least one of the three genes two distinct alleles that belonged to different groups.

DISCUSSION

LIMITS OF NUCLEAR SINGLE-COPY GENES

Amplification of all three nuclear single-copy genes was reasonably easy and consistent. Direct sequencing from the PCR products was possible for ncpGS and PHYC; when directly sequenced, GapC produced electrophoregrams with abundant polymorphisms, so cloning was always necessary.

In the absence of cytological data, we have no way to assess the existence of polyploidy in Codia. Although cases of suspected hybridization have been observed in the field, we have no evidence of their fertility and evolutionary potential. Thus, for a given accession, we could not estimate the number of alleles to expect. When direct sequencing is possible, homozygotes can clearly be distinguished from accessions with two or more alleles, but if multiple alleles exist, then cloning is necessary to tease them apart. One issue is the number of alleles to be recovered. If the direct sequence is readable, i.e. the different alleles have the same length (no net indel differences in their sequences), cloned sequences can be compared with the direct sequence, and if all alleles have been recovered, their variability should explain all polymorphisms observed in the directly sequenced electropherograms. If the direct sequence is not readable, then recovery of both alleles with more than a 95% chance of success requires the sequencing of at least six clones.

When direct sequencing is not successful, it is a priori not possible to know the number of alleles to expect. However, if one sequences six clones and recovers only a single allele, the chance that the accession is a homozygote is greater than 95% (96.875% exactly); conversely if the accession is a heterozygote, the chance that both alleles will be recovered is also more than a 95%. However, as one increases the number of accessions, the chance of missing an allele increases. When investigating two heterozygous accessions, chances of recovering both alleles from both accessions are 96.875%  96.875% ≈ 93.8%. Thus a higher chance exists for a heterozygote to be undetected. For a sample like ours (13 accessions), we would need to sequence nine clones (calculation not shown) to assure a 95% chance of detecting all heterozygotes. Thus, as one increases the number of accessions and genes, sequencing becomes time consuming and costly.

In our study, a high probability exists that some alleles for GapC were missed and some heterozygotes not detected. In contrast, we are confidant that all homozygotes for ncpGS and PHYC were detected and that by comparison with the direct sequences, most alleles in heterozygotes were probably recovered. Thus, we have given greater weight to the results obtained with ncpGS than to those obtained both with PHYC, which exhibits low variability, and with GapC, for which some alleles were probably missed.

An ideal nuclear single-copy gene would be one that can be sequenced directly so that both homozygotes and individuals with multiple alleles are readily identified. When no variation in length is found between alleles in accessions with multiple alleles, it is easier to compare the direct sequence and the cloned sequences and thus check that all alleles have been recovered. Recovering all alleles in polyploids with three or more alleles is difficult; to avoid artefacts occurring during PCR or cloning, production of multiples clones for each allele is also advantageous.

EVIDENCE FOR RETICULATE EVOLUTION IN CODIA

In networks based on ncpGS and GapC (Fig. 1), two large groups of alleles could clearly be distinguished, I and J, between which the root was connected. Two smaller allele groups, A and N, could also be distinguished. In the network based on PHYC sequences, these groups are not clearly separated due to the low level of variation in this gene, and for most of the following discussion this gene will therefore not be considered.

In our results, we observed two categories of species. Codia ferruginea, C. incrassata, C. jaffrei and C. nitida represent the first and were homozygous for all three genes or had alleles that were similar and belong to the same allele group. All other species examined, which represent the second category, exhibit for at least one of these genes two alleles that belong to different groups. A possible explanation of this pattern of variation is reticulate evolution within Codia, in which species of the second category are the result of hybridization events between species belonging to the first category. An alternative hypothesis would be incomplete lineage sorting, where, in an extreme case, most alleles would be present in most species, and homozygotes and heterozygotes would have been randomly sampled. Because a single accession was used for each species, it is not possible to infer whether the same pattern would have been observed if more individuals per species had been sampled. Nevertheless several observations suggest that hybridization, not incomplete lineage sorting, best explains our results.

First, consistent relationships between Codia ferruginea, C. incrassata and C. jaffrei observed for all three genes may be sufficient to rule out alternative hypotheses such as incomplete lineage sorting or an ancestral duplication event predating the radiation of Codia that gave rise to the modern species, which was then followed by random loss of gene copies.

Sequences of ncpGS from one additional accession of C. spatulata and of C. ferruginea, each from a location different from the one previously sampled, revealed alleles of group I and J for the first one and a single allele of the I group for the second, which is consistent with what we had already observed (Pillon, unpublished).

Heterozygotes make up a large proportion of the sampling and were also recovered in rare species such as Codia belepensis. This species is only found on a few islands off the northern coast of New Caledonia, where it is the only species of the genus present. Thus, although one might predict that this species would be fixed for a single allele through genetic drift, two distinct alleles were recovered in the ncpGS analysis.

Most heterozygotes sampled had alleles that were far apart in the networks; in the case of incomplete lineage sorting, heterozygotes would be more likely to have alleles that are similar due to more recent divergence. Furthermore, the two alleles found in such heterozygotes were generally both derived, as suggested by their being placed at the tips of networks, contrary to what would be expected in a case of incomplete lineage sorting.

Several heterozygote accessions such as those of Codia albicans, C. montana and C. triverticillata showed a consistent pattern for both ncpGS and GapC: they combined two alleles from the I and J groups in each analysis. The species that exhibit different patterns for the two genes are those displaying alleles from two distinct groups in ncpGS and a single group in GapC. However, as mentioned above, some alleles have probably been missed for this gene, especially when fewer than six clones were sequenced. In spite of this possibility, all species displaying alleles from two groups of GapC also showed alleles from two distinct groups in ncpGS, with the exception of C. mackeeana. Furthermore, in homoploid hybrid species, one parental allele is commonly lost soon after formation [START_REF] Howarth | Genealogical evidence of homoploid hybrid speciation in an adaptive radiation of Scaevola (Goodeniaceae) in the Hawaiian islands[END_REF][START_REF] Friar | Hybrid origin and genomic mosaicism of Dubautia scabra (Hawaiian silversword alliance, Asteraceae, Madiinae)[END_REF]. Thus we believe that most heterozygotes observed in this study are species of hybrid origin; this seems clear for species such as C. albicans, C. montana and C. triverticillata, which show consistent patterns for two genes, and for C. spatulata, which shows the same pattern in two accessions. It is possible that both hybridization and incomplete lineage sorting (and possibly also introgression) explain the pattern observed. The use of multiple accessions in each species would have been useful to clearly demonstrate that some species have a hybrid origin since they would be expected to be fixed heterozygotes.

Future studies will require an enlarged sampling, and due to the technical limits associated with nuclear single-copy genes discussed above, alternative markers such as AFLPs would probably be preferable. Such markers are cheaper, more easily applicable to a large sampling and provide a large number of markers spread across the genome. However their analysis is more complex and they do not necessarily allow to clearly identifying the parents of the species of hybrid origin (see e.g. [START_REF] Hedrén | Amplified fragment length polymorphisms (AFLP) reveal details of polyploid evolution in Dactylorhiza (Orchidaceae)[END_REF].

No chromosome counts are available for Codia, and thus it is not known if the species of presumed hybrid origin are homoploid or polyploid. We never found more than two alleles in any accession, although low variability of the markers used and difficulties of recovering multiple alleles in cloning make detection of more than two alleles in putative polyploids difficult. For GapC, we detected in some species of putative hybrid origin two distinct alleles corresponding to a single parental group, and thus if one assumes that the other parental allele(s) have been missed in the cloning procedure, such accessions may be polyploids.

Currently few cytological data are available for New Caledonian plants, and this study demonstrates the importance of such data. Our results are, nonetheless, more consistent with all species being diploids, but this obviously needs further investigation. GapC. In the GapC tree, it is nested in the group containing C. ferruginea and C. incrassata, whereas in the ncpGS network it is clearly distinct from the other two species; its alleles are also different from those of both the jaffrei group and the ferruginea-incrassata group by three and four substitutions, respectively. The combined analysis of the three genes suggests the following relationships: ((C. jaffrei, C. nitida)(C. incrassata, C. ferruginea)). These species are subsequently referred to as "parental species".

All other species of Codia examined exhibited alleles from two groups for at least one gene, and we therefore consider them to be species of hybrid origin (hereafter "hybrid species"). Five of them, C. albifrons, C. belepensis, C. microphylla, C. spatulata and C. triverticillata, have combinations from the I and J allele groups and can be considered the result of a cross or crosses between C. jaffrei and a member of the ferruginea-incrassata group. Codia albicans and C. montana each produced alleles belonging to two distinct groups, A and I for the former and A and J for the latter. Alleles of group A were not found in any parental lineage and may be the legacy of an extinct element that gave rise to C. albicans via a cross with a member of the ferruginea-incrassata group; it also is likely to have produced C. montana by crossing with C. jaffrei. Codia mackeeana also has alleles from both the A and I groups according to the GapC tree, but for ncpGS, no A allele was recovered. It is possible that the history of this species is more complex. Codia discolor shows peculiar patterns; for ncpGS it has a combination of alleles from the I and N groups, for PHYC it has two alleles that are similar to each another and distinct from all others, and for GapC it has one allele from the J group. The parentage suggested by ncpGS alone would be a combination of the I and N genomes, but the two other genes do not support this hypothesis. Further genes and sampling will be necessary to clarify the origin of this species. A summary of the putative evolutionary history of the genus Codia is presented in figure 3.

In the split graph produced by the combined analysis of all three loci presented in figure 2, species of presumed hybrid origin appear in between their putative parents. With the possible exceptions of Codia montana and C. albicans, it is not possible to distinguish which species are the results of hybridization events. Species with the same parentage do not form clear clusters, and their parentage is not directly interpretable from the graph. A possible weakness of POFAD is that it does not take into account missing alleles, which could be either experimental artefacts or truly the result of natural loss. Older homoploid hybrids would not be expected to exhibit both parental alleles, as demonstrated in Helianthus [START_REF] Rieseberg | Major ecological transitions in wild sunflowers facilitated by hybridization[END_REF], Scaevola [START_REF] Howarth | Genealogical evidence of homoploid hybrid speciation in an adaptive radiation of Scaevola (Goodeniaceae) in the Hawaiian islands[END_REF] and Dubautia [START_REF] Friar | Hybrid origin and genomic mosaicism of Dubautia scabra (Hawaiian silversword alliance, Asteraceae, Madiinae)[END_REF], and some of the "missing" alleles in our study may no longer be present in the putative hybrid species. Thus if one considers two species with the same parentage, one of which has lost one of the parental alleles for a given gene, these two species would not appear in exactly the same place on the tree; one will appear closer to one of the parents. The existence of an extinct parental lineage cannot be detected with this method.

MORPHOLOGY OF CODIA

Differences in reproductive characters are few in Codia, and most differences between species involve features of the leaves (especially the shape of the blade and the amount and type of indumentum) and stipules (especially size and shape; table 2). Several different patterns of character distribution are observed in hybrid taxa compared with their putative parents.

Our results show that novel characters can occur in hybrid species relative to their parents. For example, most species of Codia have opposite leaves, but two consistently have leaves in whorls of three: C. albifrons and C. triverticillata, both of which have a combination of the I and J genomes and are thus products of hybridization between opposite-leaved parents. Both also share the unique J2 allele for PHYC, suggesting they diverged from a common ancestor after the hybridization event or that their parents shared this allele. Thus, it is not clear if the appearance of this novel feature represents a single event or two parallel ones. Whorled leaves can be considered as a transgressive phenotype, comparable to what has been observed in hybrid species of Helianthus [START_REF] Rieseberg | Major ecological transitions in wild sunflowers facilitated by hybridization[END_REF][START_REF] Rieseberg | Hybridization and the colonization of novel habitats by annual sunflowers[END_REF].

Another novel character found only in species of putative hybrid origin is narrowly triangular stipules, which are found in C. discolor, C. montana and C. spatulata; each of these has a different genomic combination (Table 1), suggesting that this character has resulted from several different crosses. Examples of the converse, where characters seen in ancestral species have been lost in hybrids, also occur. For instance, C. belepensis, C. microphylla and C. spatulata all lack the pale felty indumentum present in the adult foliage of their putative parents (C. ferruginea, C. incrassata and C. jaffrei).

The occurrence of petals shows a slightly different pattern. Petals occur consistently in

Codia albifrons (I and J alleles) and C. montana (A and J alleles), and they have been observed occasionally or rarely in six other species, including the parental species C. incrassata and C. nitida. Importantly, both of the other genera in the tribe Codieae, Callicoma and Pullea [START_REF] Bradford | Phylogenetics and classification of Cunoniaceae (Oxalidales) using chloroplast DNA sequences and morphology[END_REF], have flowers that lack petals, suggesting that this could therefore be the ancestral condition for the group. Most hybrid species are restricted to one type of soil, but three occur on both (Codia albicans, C. montana and C. spatulata). The lack of sub-grouping within the I group of alleles does not allow us to distinguish whether C. ferruginea or C. incrassata is the parent of the putative hybrid species with an allele of the I group. It is also possible that these two species diverged subsequent to the hybridization events. Furthermore the ecology of the putative extinct parental lineage is not known. However, with the exception of C. albicans and C. mackeeana, at least one of the parents of each hybrid species grows on ultramafic soils. This implies that adaptation to these distinctive soils can be transmitted from parent to daughter species and can be lost, even if both parents grow on ultramafic soils.

Adaptation to ultramafic soils in Codia is possibly plesiomorphic, as de [START_REF] De Kok | Are plant adaptations to growing on serpentine soil rare or common? A few case studies from New Caledonia[END_REF] suggested for five other genera in New Caledonia. However, he used cladistic analyses based on morphological characters, which cannot detect reticulate evolution. A plesiomorphic adaptation to ultramafic soils would not be surprising if, as suggested by [START_REF] Pelletier | Geology of the New Caledonia region and its implications for the study of the New Caledonian biodiversity[END_REF], New Caledonia was entirely covered by ophiolite as it re-emerged from a period of deep-sea submersion in the late-middle Eocene (around -35My). [START_REF] Patterson | Geographic cohesion, chromosomal evolution, parallel adaptive radiations, and consequent floral adaptations in Calochortus (Calochortaceae): evidence from a cpDNA phylogeny[END_REF] found that adaptation to serpentine substrates in Californian Calochortus (Liliaceae) evolved at least seven times and was thus highly homoplasious, but again, because they used only plastid markers, cases of hybridization may have been missed.

It is clear that soil type is a significant factor in the current distribution of Codia since 10 of 13 species are restricted to either ultramafic or non-ultramafic soils. The importance of soil type in constraining distribution is strengthened by evidence of past range movements (see below), which suggests that dispersal is not a limiting factor. Fruits of Codia are small, indehiscent and woolly, and appear to be wind dispersed.

The three Codia species with broad ecological preferences, C. albicans, C. montana and C. spatulata (the last two being the most widespread members of the genus) are all putatively of hybrid origin. Codia albicans is polymorphic, and the limits between C. montana and C. spatulata are not clear in parts of their range, especially in north-western New Caledonia. It is possible that these taxa have had multiple origins, as is commonly observed for hybrid species [START_REF] Soltis | Angiosperm phylogeny inferred from multiple genes as a tool for comparative biology[END_REF], and this could help to explain their polymorphic morphology. Alternatively, these species could each have had a single origin and intermediates between them might be the result of recent hybridization in zones where they come into contact with one another, or their variation could be due to a combination of these processes.

Codia microphylla is rare and known only from a few localities in northern New Caledonia. Although herbarium specimens provide few data on its ecology, field observations suggest that this species may be restricted to phtanite, a type of siliceous sedimentary rock, that gives rise to dry, nutrient poor soils that support a short shrubby vegetation. The only other species of Codia that is sometimes found in this environment is C. montana, another hybrid species. Thus, adaptation to this novel habitat, unoccupied by any of the parental species, may have been facilitated by hybridization, as was observed in similar situations in Helianthus [START_REF] Rieseberg | Major ecological transitions in wild sunflowers facilitated by hybridization[END_REF][START_REF] Rieseberg | Hybridization and the colonization of novel habitats by annual sunflowers[END_REF].

BIOGEOGRAPHY OF CODIA

Each of the four parental species has a relatively small geographical range. Codia incrassata is restricted to the area of mica-schist and sediments of north-eastern New Caledonia, and C. ferruginea is restricted to the ultramafic strip along the central-eastern part of the island (Fig. 4). Codia jaffrei is only found in the far south, and C. nitida also occurs in this region, as well as in a few isolated localities in the north-west. The southern part of Grande Terre and much of the eastern seaboard have higher rainfall than other parts of New Caledonia (Service d'Hydrologie de l 'ORSTOM, 1981;[START_REF] Caudmont | Atlas climatique de la Nouvelle-Calédonie[END_REF], and three of the parental species are largely restricted to the wettest areas; the part of the east coast where C. ferruginea occurs is somewhat drier. Studies on palms (Pintaud & Jaffré, 2001;Pintaud et al., 2001) have suggested that the wet areas in the south and east of New Caledonia may have been refugia for rainforest taxa during the drier episodes of the Pleistocene, and the biased occurrence of diploid parental species in glacial refugia is a common feature in several polyploid complexes in Europe [START_REF] Trewick | Polyploidy, phylogeography and Pleistocene refugia of the rockfern Asplenium ceterach: evidence from chloroplast DNA[END_REF][START_REF] Abbott | History and evolution of the Arctic flora: in the footsteps of Eric Hultén[END_REF]Pillon et al., 2007). All the species of Codia occurring on the markedly drier western coast of New Caledonia have a hybrid origin, except C. nitida (see above), which is nevertheless rare there.

Further work is needed to confirm the importance of rainforest refugia in New Caledonia and to locate them precisely, and population genetics of relatively widespread species [START_REF] Taberlet | Comparative phylogeography and postglacial colonization routes in Europe[END_REF] could potentially shed some lights on this issue.

Another striking distributional feature of Codia is that the main species involved in the production of hybrids, C. jaffrei on one side and the ferruginea-incrassata group on the other, do not have overlapping ranges (Fig. 4). However, hybridization between these taxa probably took place when the parental species (the ancestors of the modern taxa) occurred in sympatry; since the origin of the hybrids, the ranges of the parental taxa must have shifted, leaving their modern descendents in allopatry. Similar shifts in ranges of the parents of hybrids are thought to have occurred in Nicotiana [START_REF] Chase | Molecular systematics, GISH and the origin of hybrid taxa in Nicotiana (Solanaceae)[END_REF]. Transgressive hybrids are well known for their altered ranges relative to their parental taxa. Hybridization events that have given rise to most species of Codia were thus probably relatively ancient, and their current distributions may not be representative of earlier points in their history. We have little information about alterations in climate and vegetation types between the emergence of New Caledonia in the late Eocene [START_REF] Pelletier | Geology of the New Caledonia region and its implications for the study of the New Caledonian biodiversity[END_REF] and today, but changes in Quaternary vegetation have been charted by palynological studies [START_REF] Stevenson | A late Quaternary record of environmental change and human impact from New Caledonia[END_REF][START_REF] Stevenson | A comparison of late Quaternary forest changes in New Caledonia and northeastern Australia[END_REF][START_REF] Cabioch | Evolution des paléoenvironnements dans le Pacifique lors de la dernière déglaciation : exemples en Nouvelle-Calédonie et au Vanuatu[END_REF].

Further evidence of alterations in distribution comes from hybrid species that have restricted distributions and do not co-occur with their parents; these include Codia belepensis, C. microphylla and C. triverticillata. Codia belepensis is an extreme case as it is restricted to three islands off the north of Grande Terre, Yandé, Art and Pott, where it is the only member of the genus present. The two species with whorled leaves, C. albifrons and C. triverticillata, which are possibly descended from the same hybrid ancestor, do not co-occur at present; the former is restricted to the south of New Caledonia, and the other is found on only three mountains near the north-western coast: Kopeto, Boulinda and Paéoua.

Considerable range alterations, such as those we have hypothesised to have occurred in Codia, have be detected in other genera such as Gossypium, in which [START_REF] Cronn | Cryptic repeated genomic recombination during speciation in Gossypium gossypioides[END_REF] found evidence of introgression in the Mexican cotton G. gossypioides from an African species. Similarly, all species of Nicotiana sect. Suaveolentes [START_REF] Chase | Molecular systematics, GISH and the origin of hybrid taxa in Nicotiana (Solanaceae)[END_REF], restricted to Africa (Namibia) and Oceania and comprising ca. 25 species, are the result a single hybridisation involving parents from two lineages that are now confined to South America. [START_REF] Barraclough | Detecting the geographical pattern of speciation from species-level phylogenies[END_REF] suggested using the level of range overlap between sister species to determine the dominant mode of speciation: sympatric or allopatric, but when applied to South African Protea, [START_REF] Barraclough | The causes of speciation in flowering plant lineages: species-level DNA trees in the African genus Protea. In: Plant species-level systematics: new perspectives on pattern and process[END_REF] found evidence for extensive range movements of species, preventing them from determining the mode of speciation in this genus. Hybrid speciation has been important in Codia, despite what contemporary species distributions suggest. Range changes could be a common and important phenomenon that obscures both biogeographic patterns and the historical diversification of a clade.

IMPLICATIONS FOR CONSERVATION

New Caledonian biodiversity is currently facing multiple threats, including fires, nickel mining, invasive species and climate change. The small size of the Archipelago (18,500 km 2 ) and importance of micro-endemism almost certainly mean that a large proportion of New Caledonian species are susceptible to extinction and should be included in the IUCN Red List, the updating of which is urgently needed for New Caledonia [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]. Prioritisation of species and areas to preserve is needed, especially as few ex-situ conservation programs currently exist for threatened species in New Caledonia. [START_REF] Vane-Wright | What to protect?─Systematics and the agony of choice[END_REF] suggested that some species have higher conservation value than others, i.e. they have no close relatives. For example, the two recently described species, Codia jaffrei and C. triverticillata [START_REF] Hopkins | Four new species in the endemic genus Codia (Cunoniaceae) from New Caledonia[END_REF], are both directly threatened by mining activities and have been assigned the provisional IUCN status of vulnerable (VU) and endangered (EN), respectively. Based on the level of threat, conservation priority might be given to the more threatened species, C. triverticillata. However, C. jaffrei represents a distinct parental lineage, whereas C. triverticillata is a hybrid species with a genomic content similar to that of several other species, e.g. C. albifrons and C. belepensis. The evolutionary potential of C. jaffrei may therefore be considered higher as it might hybridize again with other parental species to give rise to new hybrid taxa, and thus, it could be argued that C. jaffrei is of greater conservation importance than C. triverticillata.

The results of this study support the idea of Pintaud et al. (2001) and Pintaud & Jaffré (2001) that the southern and eastern parts of New Caledonia may have served as rainforest refugia during the climatic oscillations of the Pleistocene. Southern New Caledonia is the most species-rich region for Cunoniaceae; in addition, of the five families endemic to the Archipelago (Amborellaceae, Oncothecaceae, Paracryphiaceae, Phellinaceae and Strasburgeriaceae), all but Amborellaceae occur there, and two (Oncothecaceae and Strasburgeriaceae) are restricted to this area. Refugia are also expected to host greater genetic diversity [START_REF] Taberlet | Comparative phylogeography and postglacial colonization routes in Europe[END_REF] and more rare genotypes [START_REF] Petit | Glacial refugia: hotspots but not melting pots of genetic diversity[END_REF] within species and to represent important areas for the long-term persistence of biodiversity [START_REF] Taberlet | Quaternary refugia and persistence of biodiversity[END_REF]. Thus, these possible refugia should be given greater conservation attention, the network of southern reserves should be re-enforced and new reserves should be designated on the eastern coast where there are currently almost none [START_REF] Jaffré | Threatened plants of New Caledonia: is the system of protected areas adequate?[END_REF].

It has been argued that it is important to preserve the conditions required for the persistence of evolutionary processes that generate biodiversity [START_REF] Smith | The preservation of process: the missing element of conservation program[END_REF][START_REF] Cowling | Rapid plant diversification: planning for an evolutionary future[END_REF][START_REF] Moritz | Strategies to protect biological diversity and the evolutionary processes that sustain it[END_REF]. This could be a particularly appropriate strategy in the case of species complexes that are evolving rapidly and/or where species delimitations are controversial [START_REF] Ennos | Conserving taxonomic complexity[END_REF][START_REF] Pillon | Species diversity versus phylogenetic diversity: a practical study in the taxonomically difficult genus Dactylorhiza (Orchidaceae)[END_REF]. In New Caledonia, most plant species are restricted to one type of substrate, either ultramafic or non-ultramafic, and zones between these two soil types that still have intact vegetation are not numerous, but they provide evolutionarily important opportunities for some species to co-occur. One such site is Mont Koghis, close to the capital city of Nouméa, where rainforest covers a complex matrix of soils derived from peridotite, serpentine, and sedimentary rocks, and species that are not normally sympatric due to divergent ecological preferences are found in close proximity. It appears likely that hybridization between species with different substrate preferences has given rise to Cunonia koghicola on this mountain [START_REF] Pillon | Two new species of Cunonia (Cunoniaceae) from New Caledonia[END_REF]. Such sites should be preserved to maintain one of the evolutionary processes that have contributed to the remarkable biodiversity of New Caledonia. Rare occurrences are in parentheses.

1 UM = ultramafic, NUM = non-ultramafic.

2 F = forest, M = maquis and/or other types of scrub.

  Five species, C. albifrons, C. belepensis, C. microphylla, C. spatulata and C. triverticillata, displayed a combination of alleles from groups I and J. Codia albicans and C. mackeeana had a combination of alleles from groups A and I, and C. montana a combination of A and J. Codia discolor produced alleles of the J group for PHYC and GapC and alleles from groups I and N for ncpGS. A split-graph showing relationships of the Codia species is shown in figure 2. Little structure is observed, but species that combine I and J alleles cluster between C. incrassata and C. jaffrei.

  EVOLUTIONARY HISTORY OF CODIA Four species of Codia did not show signs of hybridization in any of the analyses: C. ferruginea, C. incrassata, C. jaffrei and C. nitida. All three genes indicate a close relationship between C. ferruginea and C. incrassata and show that C. jaffrei is more distantly related (Fig. 1). The placement of C. nitida was somewhat different in the analyses of ncpGS and

  ECOLOGY OF CODIAThe preferred soil types (ultramafic or non-ultramafic) of Codia species are summarised in table 2, together with an indication of habitat, which can include open scrub and maquis, other disturbed situations such as roadsides and along tracks, and forest edges and interiors. Although some species have restricted ecological preferences, several show a degree of plasticity.The four parental species with no evidence of hybridization are each restricted to a single soil type: Codia ferruginea, C. jaffrei and C. nitida only grow on ultramafic soils, and C. incrassata occurs only on non-ultramafic substrates. Thus, adaptation to ultramafic soils appears either to be ancestral for the genus and to have been lost in C. incrassata, or it may have evolved twice, in the C. jaffrei-C. nitida clade and in C. ferruginea.

  3 A = triangular, broadly ovate or cordate ; B = narrowly triangular ; C = elliptic.

Figure 1 .

 1 Figure 1. Network of alleles obtained with ncpGS, PHYC and GapC.

Figure 2 .Figure 3 .

 23 Figure 2. Split network of the species of Codia based on all three genes (ncpGS, PHYC and

Figure 4 .

 4 Figure 4. Distributions of the three parental species C. ferruginea, C. incrassata and C. jaffrei

Table 1 .

 1 List of alleles found in each Codia accession and their supposed genomic composition. Uncertain genomic compositions are in parenthesis. For GapC, the number of clones successfully sequenced is indicated in parenthesis.

	Species	ncpGS	PhyC	GapC	Genome
	C. albicans	A2,I10	A1,J1	A1,I10 (4) A,I
	C. albifrons	I3,J3	J2	I1,I8 (5)	I,J
	C. belepensis	I8,J6	J3	I2,I4 (4)	I,J
	C. discolor	I5,N3	J4,J5	J2 (5)	(I,N)
	C. ferruginea	I4	I2	I9,I11 (3)	I
	C. incrassata	I2	I3	I7 (3)	I
	C. jaffrei	J1	J1	J1 (6)	J
	C. mackeeana	I1,I9	I1,J1	A3,I3 (6)	A,I
	C. microphylla	I6,J4	-	J4,J5 (6)	I,J
	C. montana	A1,J7	A1	A2,J6 (4)	A,J
	C. nitida	N1,N2	J1	N1 (4)	N
	C. spatulata	I7,J5	I1	I5 (1)	I,J
	C. triverticillata	I1,J2	J2	I6,J3 (5)	I,J

Table 2 .

 2 Ecology and morphology of Codia species.

							pale,	felty	arachnoid	hairs	stipules	3	petals
	Species	substrate 1	habitat	2	node # leaves per	lower leaf surface indumentum on	on leaves
	C. albicans	UM	NUM	(F)	M	2	+		--		A	--
	C. albifrons	UM		(F)	M	3	+		++		A	++
	C. belepensis	UM			M	2	--		--		A	--
	C. discolor	UM		(F)	M	2	+		--		B	(+)
	C. ferruginea	UM			M	2	+		+		A	(+)
	C. incrassata		NUM	F	(M)	2	+		--		A	--
	C. jaffrei	UM		F		2	+		--		A	(+)
	C. mackeeana		NUM	F		2	--		--		A	--
	C. microphylla		NUM		M	2	--		(+)		A-B	(+)
	C. montana	UM	NUM	(F)	M	2	--		--		B	++
	C. nitida	UM		(F)	M	2	--		--		C	(+)
	C. spatulata	UM	NUM	F	M	2	--		--		B	(+)
	C. triverticillata UM			M	3	+		--		A	--

  Supplementary data. List of Codia accessions included in the study with voucher information and GenBank accessions number for DNA sequences.

	Species	Voucher	Herbarium Locality	ncpGS	PhyC	GapC
	Codia albicans Vieill. ex Pamp.	Pillon et al. 478	NOU	Hienghène	FJ595023 FJ595024	FJ595046 FJ595047	FJ595061 FJ595062
	Codia albifrons (Brongn. ex Schinz & Guillaumin) Baker f. Bradford & Hopkins 1144	MO,NOU Koghis	FJ595025 FJ595026	FJ595048	FJ595063 FJ595064
	Codia belepensis H.C.Hopkins	Munzinger & Swenson 3031	NOU	Yandé	FJ595027 FJ595028	FJ595049	FJ595065 FJ595066
	Codia discolor (Brongn. & Gris) Guillaumin	Bradford et al. 1193	MO,NOU Thio	FJ595029 FJ595030	FJ595050 FJ595051	FJ595067
	Codia ferruginea Brongn. & Gris	Pillon & Barrabé 330	NOU	Cap Bocage FJ595031 FJ595052	FJ595068 FJ595069
	Codia incrassata Pamp.	Pillon & Munzinger 80	K,P,NOU Mandjélia	FJ595032 FJ595053 FJ595070
	Codia jaffrei H.C.Hopkins & Fogliani	Pillon 38	NOU	Grand Kaori FJ595033 FJ595054 FJ595071
	Codia mackeeana H.C.Hopkins & Fogliani	Fogliani & Hopkins 120 [Type] K,P,NOU Col d'Amieu	FJ595034 FJ595035	FJ595055 FJ595056	FJ595072 FJ595073
	Codia microphylla Vieill. ex Guillaumin	Munzinger 2956	NOU	Col d'Arama	FJ595036 FJ595037	-	FJ595074 FJ595075
	Codia montana J.R.Forst. & G.Forst.	Pillon & Vergnes 225	NOU	Tao	FJ595038 FJ595039	FJ595057	FJ595076 FJ595077
	Codia nitida Schltr.	Bradford & Hopkins 1149	MO,NOU Koghis	FJ595040 FJ595041	FJ595058 FJ595078
	Codia spatulata Brongn. & Gris	Pillon 158	NOU	Lac de Yaté	FJ595042 FJ595043	FJ595059 FJ595079
	Codia triverticillata H.C.Hopkins & Pillon	Pillon et al. 137	K,P,NOU Kopeto	FJ595044 FJ595045	FJ595060	FJ595080 FJ595081

ACKNOWLEDGMENTS

The authors wish to thank the Direction de l'Environnement (Province Sud) and DDEE (Province Nord) for permitting scientific collections in New Caledonia. We also thank all staff and students of the Botanical Laboratory at IRD, Alan Vergnes and Hervé Jourdan for assistance in the field. Jason Bradford also kindly provided some leave 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, Maria Vorontsova, Nina Rønsted, and Jim Clarkson for discussions on nuclear single-copy genes. Anaïs Cardineau and Cyril Poullain (CNRS) provided a Macintosh computer to complete data analysis. We thank Pete Lowry and one anonymous reviewer for comments on an earlier version of the manuscript.