Anne Duputié
email: anne.duputie@ens-lyon.org

Patrice David

Chantal Debain

Doyle Mckey

Natural hybridization between a clonally propagated crop, cassava (Manihot esculenta Crantz) and a wild relative in French Guiana

Keywords: Biosafety, cassava, clonal propagation, morphological variation, natural hybridisation, wild relative

come   L'archive ouverte pluridisciplinaire

Because domestication rarely leads to speciation, domesticated populations often hybridise with wild relatives when they occur in close proximity. Little work has focused on this question in clonally propagated crops. If selection on the capacity for sexual reproduction has been relaxed, these crops would not be expected to hybridise with their wild relatives as frequently as seed-propagated crops. Cassava is one of the most important clonally-propagated plants in tropical agriculture. Gene flow between cassava and wild relatives has often been postulated, but never demonstrated in nature. We studied a population of a wild Manihot sp. in French Guiana, which was recently in contact with domesticated cassava, and characterized phenotypes (ten morphological traits) and genotypes (six microsatellite loci) of individuals in a transect parallel to the direction of hypothesized gene flow. Wild and domesticated populations were strongly differentiated at microsatellite loci. We identified many hybrids forming a continuum between these two populations, and phenotypic variation was strongly correlated with the degree of hybridisation as determined by molecular markers. Analysis of linkage disequilibrium and of the diversity of hybrid pedigrees showed that hybridisation has gone on for at least three generations and that no strong barrier prevents admixture of the populations. Hybrids were more heterozygous than either wild or domesticated individuals, and phenotypic comparisons suggested heterosis in vegetative traits. Our results also suggest that this situation is not uncommon, at least in French Guiana, and demonstrate the need for integrated management of wild and domesticated populations even in clonally propagated crops.

Hybridisation is considered to be a major evolutionary process [START_REF] Arnold | Are natural hybrids fit or unfit relative to their parents?[END_REF][START_REF] Ellstrand | Gene flow and introgression from domesticated plants into their wild relatives[END_REF][START_REF] Rieseberg | Major ecological transitions in wild sunflowers facilitated by hybridisation[END_REF]. It is a primordial issue for many crop species because plant domestication has generally not led to total reproductive isolation of domesticated and wild populations [START_REF] Ellstrand | Gene flow and introgression from domesticated plants into their wild relatives[END_REF]. Hybridisation affects the functioning of both kinds of populations [START_REF] Ellstrand | Gene flow and introgression from domesticated plants into their wild relatives[END_REF]Gepts & Papa 2003), and can lower or increase the degree of adaptation in each. Because domestication often involved bottlenecks and loss of genetic diversity, gene flow from wild to domesticated taxa can be agronomically useful, favouring local adaptation and increasing genetic diversity, thereby lowering risks of crop failure due to variation in biotic or abiotic environmental factors [START_REF] Jarvis | Wild relatives and crop cultivars: detecting natural introgression and farmer selection of new genetic combinations in agroecosystems[END_REF]. The crop's close relatives are thus considered important genetic resources, as a reservoir of diversity from which breeding programs can introgress traits into recipient cultivars. On the other hand, introgression from domesticated plants may lower the fitness and jeopardize the persistence of wild populations often already threatened by habitat loss (e.g., [START_REF] Ellstrand | Gene flow and introgression from domesticated plants into their wild relatives[END_REF]. Introgression sometimes results in genetic swamping of the wild taxon or even in its extinction by hybridisation (Gepts & Papa 2003). Whatever the direction of gene flow, hybrid fitness can differ substantially from that of parental taxa. Where parental populations show very divergent adaptations, reduced hybrid fitness can reinforce reproductive isolation [START_REF] Barton | Analysis of hybrid zones[END_REF]. Alternatively, hybrids can show heterosis, i.e. increased vigour, and sometimes become invasive weeds (reviewed by [START_REF] Ellstrand | Gene flow and introgression from domesticated plants into their wild relatives[END_REF]. They may also be fitter than their parents in some environments, which can ultimately lead to hybrid speciation (e.g., [START_REF] Rieseberg | Major ecological transitions in wild sunflowers facilitated by hybridisation[END_REF]. Hybrid vigour depends on both genetic [START_REF] Arnold | Are natural hybrids fit or unfit relative to their parents?[END_REF] and environmental factors (Rieseberg et al. 2003). Joint management of crops and their wild relatives thus appears to be a necessity in primary centres of diversity. Achieving it requires prior knowledge of their ability to hybridise in nature, and characterization of CROP-WILD NATURAL HYBRIDIZATION IN CASSAVA the environments in which introgression can occur. With the notable exceptions of potato [START_REF] Celis | Environmental biosafety and transgenic potato in a centre of diversity for this crop[END_REF]) and yam [START_REF] Scarcelli | Farmers' use of wild relative and sexual reproduction in a vegetatively propagated crop. The case of yam in Benin[END_REF], relatively little attention appears to have been focused on this theme in clonally propagated crops. Yet, these may differ from sexually propagated crops because selection may have favoured traits that result in reduced gene flow from or towards their wild relatives. First, clonal propagation is expected to relax selection on traits involved in sexual reproduction (seeds and pollen) and could therefore decrease opportunities for pollen or seed flow. However, clonal propagation may not exclude the occasional or regular incorporation of individuals from seeds (and thus potential hybrids). The importance of sexual reproduction in populations of these plants has often been underestimated (e.g., Pujol et al. 2005a[START_REF] Pujol | Size asymmetry in intraspecific competition and the density-dependence of inbreeding depression in a natural plant population: a case study in cassava (Manihot esculenta Crantz, Euphorbiaceae)[END_REF]. Second, clonally propagated plants benefit from fixed favourable gene combinations. Hence crop-wild hybrids might have lower agronomic interest, and thus lower fitness in domesticated populations, than established clones. There are thus many open questions about the extent to which clonally propagated crops hybridise with their wild relatives, and the effects of hybridization on vigour and fitness. Potato [START_REF] Celis | Environmental biosafety and transgenic potato in a centre of diversity for this crop[END_REF]) and yam [START_REF] Scarcelli | Farmers' use of wild relative and sexual reproduction in a vegetatively propagated crop. The case of yam in Benin[END_REF] can hybridise in the field with several wild relatives, but neither F1 vigour nor F1 fertility have been studied. In this study, we focused on cassava (Manihot esculenta Crantz), a crop propagated clonally by stem cuttings. Its domestication syndrome includes the thickening of stems, achieved in part through reduction in the extent of branching [START_REF] Jennings | Cassava[END_REF]. As in all Manihot species, branching is strictly associated with flowering (e.g., [START_REF] Médard | Morphogenèse du manioc, Manihot esculenta Crantz (Euphorbiacées-Crotonoïdées): étude descriptive[END_REF]; clonal propagation and sexual reproduction are thus in direct competition [START_REF] Jennings | Cassava[END_REF]. Cassava therefore typifies a crop where clonal propagation may have resulted in decreased opportunity for hybridisation with wild relatives. This view was recently challenged when we identified, on the basis of morphological traits, a putative hybrid zone between cassava and one of its wild relatives in French Guiana. Using data from six microsatellite markers, we assess the reality of hybridisation, evaluate whether the phenomenon is frequent, whether it is ephemeral or long-lasting, and discuss conservation issues arising from our results. The question of crop/wild gene flow acquires an additional interesting dimension as some field trials of transgenic cassava varieties have already been conducted, and others are planned.

MATERIALS AND METHODS

Cassava biology / study model

The genus Manihot, last revised over 30 years ago [START_REF] Rogers | Manihot, Manihotoides (Euphorbiaceae). Flora Neotropica[END_REF], includes 98 currently recognized species, although this number should probably be revised downward [START_REF] Second | Molecular marker (AFLP)-based Manihot and cassava numerical taxonomy and genetic structure analysis in progress: Implications for their dynamic conservation and genetic mapping[END_REF]. Domesticated cassava was already present in Panama 5,000-7,000 years ago [START_REF] Piperno | Starch grains reveal early root crop horticulture in the Panamanian tropical forest[END_REF] but the plant was probably domesticated even earlier, as genetic data tend to show that the centre of origin of the crop lies along the southern rim of the Amazon basin [START_REF] Olsen | Evidence on the origin of cassava: Phylogeography of Manihot esculenta[END_REF][START_REF] Léotard | Phylogéographie et origine de la domestication du manioc (Manihot esculenta Crantz, Euphorbiaceae): les apports d'un échantillon élargi à l'écotone nord de l'Amazonie[END_REF][START_REF] Olsen | SNPs, SSRs and inferences on cassava's origin[END_REF]. It is still unclear whether cassava is a "compilospecies" derived from multiple hybridisation events [START_REF] Rogers | Manihot, Manihotoides (Euphorbiaceae). Flora Neotropica[END_REF][START_REF] Nassar | Gene flow between cassava, Manihot esculenta Crantz, and wild relatives[END_REF], or has a single ancestor, Manihot esculenta ssp. flabellifolia (Pohl) Ciferri, the latter hypothesis being better supported by currently available molecular evidence [START_REF] Olsen | Evidence on the origin of cassava: Phylogeography of Manihot esculenta[END_REF][START_REF] Elias | Assessment of genetic variability in a traditional cassava (Manihot esculenta Crantz) farming system, using AFLP markers[END_REF][START_REF] Olsen | SNPs, SSRs and inferences on cassava's origin[END_REF]). The role of postdomestication gene flow in either direction is still unclear [START_REF] Fregene | Variability of chloroplast DNA and nuclear ribosomal DNA in cassava (Manihot esculenta Crantz) and its wild relatives[END_REF]. Work by plant breeders has shown that cassava can be crossed with numerous wild congeners [START_REF] Jennings | Manihot melanobasis Müll. Arg.-a useful parent for cassava breeding[END_REF][START_REF] Wanyera | Introgression of ceara rubber (Manihot glaziovii Muell-Arg) into cassava (Manihot esculenta Crantz): a morphological and electrophoretic evidence[END_REF][START_REF] Nassar | Gene flow between cassava, Manihot esculenta Crantz, and wild relatives[END_REF]). There is no evidence for cytogenetic barriers, as all species studied so far share 2n = 36 [START_REF] Rogers | Manihot, Manihotoides (Euphorbiaceae). Flora Neotropica[END_REF]. Also, there is no evidence for strong phenological barriers. Growth, branching, and hence flowering, are all spread over several months, so that flowering periods of cassava and its wild relatives overlap broadly. Nevertheless, cassava shows a lower degree of flowering than its wild relatives, as expected for a clonally propagated crop. Cassava has inherited from its wild ancestor(s) a set of adaptations to the periodically disturbed environments they typically inhabit (Pujol et al. 2005b). These adaptations continue to function in slash-and-burn agroecosystems. Seeds are buried by ants and remain dormant when a parcel is left to fallow; they germinate when the vegetation cover of the parcel is slashed and burned [START_REF] Elias | Unmanaged sexual reproduction and the dynamics of genetic diversity of a vegetatively propagated crop plant, cassava (Manihot esculenta Crantz), in a traditional farming system[END_REF]Pujol et al. 2005b). In traditional Amerindian farming systems, farmers regularly incorporate seedlings, injecting new products of sex into their stocks of clones [START_REF] Elias | Unmanaged sexual reproduction and the dynamics of genetic diversity of a vegetatively propagated crop plant, cassava (Manihot esculenta Crantz), in a traditional farming system[END_REF][START_REF] Pujol | Size asymmetry in intraspecific competition and the density-dependence of inbreeding depression in a natural plant population: a case study in cassava (Manihot esculenta Crantz, Euphorbiaceae)[END_REF]. Cassava has long been cultivated in French Guiana. In historical times it has usually been grown in shiftingcultivation settings in forest clearings. In such settings, it is unlikely to be in proximity to its close wild relatives, which are found in savannas. Recently, however, small opportunistic plantings of a few 'sticks' of cassava are seen with increasing frequency in savanna settings, for example along drainage ditches or irrigation canals, where they sometimes are near populations of a wild relative. This wild relative, widespread in the Guianas, is locally abundant in coastal savannas of French Guiana and also occurs in savanna islands ("savanes-roches") on inselbergs in the densely forested southern part of this overseas French department. Its taxonomic identity is not yet clear. [START_REF] Allem | The primary gene pool of cassava (Manihot esculenta Crantz subspecies esculenta, Euphorbiaceae)[END_REF] considered it to belong to M. esculenta ssp. flabellifolia Pohl, but data on both microsatellite markers and G3pdh haplotypes [START_REF] Léotard | Phylogéographie et origine de la domestication du manioc (Manihot esculenta Crantz, Euphorbiaceae): les apports d'un échantillon élargi à l'écotone nord de l'Amazonie[END_REF] place it closer to M. pruinosa Pohl. However, its morphology does not match that of specimens of M. pruinosa nor of M. esculenta ssp. flabellifolia that we have examined in several herbaria. Natural clonal propagation has not been reported for this species. [START_REF] Léotard | Phylogéographie et origine de la domestication du manioc (Manihot esculenta Crantz, Euphorbiaceae): les apports d'un échantillon élargi à l'écotone nord de l'Amazonie[END_REF].

Sampling

Sampling was conducted in February 2004 along a 75m long, 15-m wide transect parallel to the hypothesized direction of gene flow between domesticated and wild individuals. Within each 5x5 m quadrat, we mapped and collected a few young leaves from all sexually mature plants and ten randomly chosen juveniles (Figure 1). We defined sexually mature plants as those that had branched at least once [START_REF] Médard | Morphogenèse du manioc, Manihot esculenta Crantz (Euphorbiacées-Crotonoïdées): étude descriptive[END_REF]. Overall, 711 individuals (468 adults and 243 juveniles) were sampled in Savane Manuel.

Because the domesticated plants supposed to be at the origin of the hybridisation events had been removed since February 2003, we sampled domesticated cassava plants in a few farmers' fields near Savane Manuel, in order to determine the alleles present in the local domesticated pool. We thus collected the greatest possible diversity of varieties (landraces). Each variety is phenotypically distinct and genetically differentiated from others, and corresponds to a clone or (more often) a set of clones [START_REF] Elias | Assessment of genetic variability in a traditional cassava (Manihot esculenta Crantz) farming system, using AFLP markers[END_REF][START_REF] Elias | Unmanaged sexual reproduction and the dynamics of genetic diversity of a vegetatively propagated crop plant, cassava (Manihot esculenta Crantz), in a traditional farming system[END_REF]. Farmers recognise varieties as different entities and manage them as such, purposely planting various assemblages of varieties within each field. A total of 69 cassava accessions, belonging to 27 varieties, were collected from the fields of four farmers.

To assess whether WM individuals from Savane Manuel were truly wild, we included in the analysis 100 wild individuals from populations where domesticated cassava was absent and no hybridisation was suspected. They belonged to twelve populations from the coastal area (from 90 km south to 60 km north of Sinnamary) and two inselberg populations. Four to ten individuals were genotyped in each population.

Collection of morphological data

For each adult individual sampled, we recorded data for the following morphological traits: number of stems from the rootstock, basal diameter of each stem, total height of the plant, height at first branching, number of leaf lobes, length and width of the central lobe of the largest leaf of the plant, petiole colour (four classes: green, pink, red, purple), stipule colour (four classes: green, pink to red, brownish, and purple) and stipule persistence (coded in four classes from highly deciduous to highly persistent). In addition, all individuals were assigned to one of four morphotypes: domesticated (DM) for the plants found in cultivation, and wild (WM), intermediate (IM) and juvenile (JM) for those from the transect, as judged by visual assessment of the overall phenotype ("Gestalt") in the field. Wild or domesticated status cannot be judged from morphology in juvenile individuals. Morphological data were not collected for the wild individuals outside Savane Manuel.

Genotyping

All individuals were genotyped for six microsatellite loci: GA12, GA57, GA126 and GA127 (Chavarriaga-Aguirre et al. 1998), and SSR55 and SSR68 [START_REF] Mba | Simple sequence repeat (SSR) markers survey of the cassava (Manihot esculenta Crantz) genome: towards an SSR-based molecular genetic map of cassava[END_REF]. All of these are on different linkage groups [START_REF] Mba | Simple sequence repeat (SSR) markers survey of the cassava (Manihot esculenta Crantz) genome: towards an SSR-based molecular genetic map of cassava[END_REF]. The allopatric wild individuals were genotyped at all but one locus (GA127). DNA was extracted using DNeasy® 96 Plant kit (Qiagen GmbH, Hilden, Germany). PCR amplification was performed in a multiplex reaction, in a final volume of 10 µL, using 3 µL of diluted DNA in each reaction. Amplification was conducted over 30 amplification cycles with an annealing temperature of 55°C.

Locus GA127 was amplified apart from the others, in a final volume of 12 µL, using 0.2 U Taq polymerase, 2.5 µM MgCl2, 125 µM each nucleotide, 0.4 µM of each primer (all products from Eurogentec, Seraing, Belgium). Annealing took place at 45°C. All amplification reactions were performed in a PTC-100™ thermocycler (MJ Research, Watertown, MA, USA) and genotyping was conducted in POP4 polymer on ABI sequencers. Genotypes were then analysed using GENESCAN ANALYSIS v.3.1.2 and GENOTYPER 2.5 (all products, instruments and software from Applied Biosystems, Foster City, CA, USA).

Analysis of population genetic structure

Allelic richness, pairwise linkage disequilibria, RST, f and θ were calculated and tested using FSTAT version 2.9.3.2 [START_REF] Goudet | FSTAT (Version 1.2): A computer program to calculate F-statistics[END_REF]. RST is a between-population differentiation coefficient, analogous to FST, which takes into account allelic sizes of microsatellites [START_REF] Slatkin | A measure of population subdivision based on microsatellite allele frequencies[END_REF]. f and θ are unbiased estimates of FIS and FST (respectively), derived by [START_REF] Weir | Estimating F-statistics for the analysis of population structure[END_REF].

Observed and expected heterozygosities were computed using GENEPOP version 1.2 (Raymond &

CROP-WILD NATURAL HYBRIDIZATION IN CASSAVA

Rousset 1995). First, we determined whether WM individuals were representative of the wild taxon. We used the STRUCTURE software (version 2.1; [START_REF] Pritchard | Inference of population structure using multilocus genotype data[END_REF], which relies on a Bayesian method that clusters similar multilocus genotypes. We used the "admixture" option, which infers, for each individual, the proportion of its genes coming from each cluster. Setting the number of clusters to two, this procedure is theoretically relevant to the study of hybridisation, as it allows, without prior information, to identify two sets of allele frequencies typical of parental populations, and the hybrid index within each individual. We first ran it including all individuals from Savane Manuel (IM, JM and WM), DM and the allopatric wild individuals (Table 2, run 1), using data for the five loci that had been sampled for all populations. As WM individuals clustered with the allopatric wild samples, meaning that WM individuals were representative of the wild taxon, we then dropped the allopatric samples from further analyses, in order to use data from all six loci. We then ran the program allowing for the formation of two clusters (Table 2, run 2). Finally, we ran the program allowing three clusters (Table 2, run 3), to evaluate the possibility that intermediate morphotypes are a completely different group, rather than being hybrids. Each run comprised a burn-in period of 200 000 iterations, followed by 10 6 MCMC steps, and was repeated two further times to confirm the repeatability of the result. We considered that an individual was "pure X" when its inferred proportion of genes belonging to cluster X exceeded 90%. With K = 2, all domesticated individuals were inferred to be "pure A" and most of the WM "pure B" (see Results). Allelic frequencies were very different in groups A and B.

Population genetic differentiation was illustrated by a factorial correspondence analysis (FCA), which was conducted using GENETIX version 4.03 [START_REF] Belkhir | GENETIX 4.03, Logiciel Sous Windows™ Pour la Génétique Des Populations[END_REF]. This analysis discriminated wild and domesticated individuals (see Fig. 2). Alleles were clustered in two groups according to their PC1 coordinate. We defined two synthetic alleles: synthetic allele W, characteristic of population WM, and synthetic allele D, characteristic of DM individuals. Synthetic alleles D and W gathered all alleles with PC1 > 0 and PC1 < 0, respectively. They corresponded to the alleles inferred by STRUCTURE to be characteristic of clusters A and B, respectively. For each individual, hybrid index was defined as its proportion of W synthetic allele. Synthetic alleles were further used in computing between-genome (κ1,1, deviation from Hardy-Weinberg equilibrium) and within-genome (κ0,2, linkage disequilibrium) pairwise associations, following the method described by [START_REF] Barton | Estimating multilocus linkage disequilibria[END_REF], using MATHEMATICA 3.0 (Wolfram Research, Oxford, UK) add-ons provided by Barton (http://helios.bto.ed.ac.uk/evolgen/barton/index.html) and modified for 6 loci. This method estimates multilocus linkage disequilibrium and Hardy-Weinberg disequilibrium in the whole sample. Different nested models were tested for their likelihood, as advised by [START_REF] Barton | Estimating multilocus linkage disequilibria[END_REF]. We first assumed a model with both Hardy-Weinberg and linkage equilibrium, then models allowing within-genome associations, both betweengenome and within-genome associations, and finally also higher-order associations, assuming first equivalent, then different contributions of each locus to the multilocus association. The most likely model was then chosen by applying the likelihood ratio test, and a 95% confidence interval was computed carrying out a random walk algorithm with T initially set to 1, as recommended by [START_REF] Barton | Estimating multilocus linkage disequilibria[END_REF]. κ1,1 and κ0,2 were computed over all six loci and including all 668 individuals. The program was also run for the adult (IM + WM subsamples) and juvenile (JM) subsets.

Analysis of morphological data

We analysed all morphological information, including both quantitative and qualitative variables, through a generalized PCA, as described by [START_REF] Hill | Principal component analysis of taxonomic data with multi-state discrete characters[END_REF], performed with ADE-4 [START_REF] Thioulouse | ADE-4: a multivariate analysis and graphical display software[END_REF]. This analysis allows for equal weighting of the qualitative and quantitative variables, whatever the number of modalities used in coding the qualitative variables.

Inter-population genetic structure

Basic statistics Differentiation among morphotypes DM, IM and WM was high: θ = 0.183 (0.130 -0.262). RST was also high: RST = 0.217, meaning that these three morphotypes were genetically differentiated, not only in terms of allele frequencies, but also of allele sizes.

At each of the six loci, allelic frequencies were very different in each morphotype (Fig. 3). Most alleles were diagnostic or quasi-diagnostic between wild and domesticated morphotypes (61% of them were present at frequencies <1% in one or the other of these two groups

Characterization of WM individuals

Further analysis using STRUCTURE software and including all individuals and all five loci genotyped for the allopatric wild individuals, with no prior population information and allowing the formation of two clusters, led to a clear clustering of DM individuals in cluster A (100% of them were 'pure A') and of allopatric wild individuals, together with WM individuals, in cluster B (89.0% and 66.9% of them, respectively, were 'pure B') (Table 2, run 1). Average posterior probability of belonging to cluster B was 85.9% for WM individuals. 33.6% of the juveniles also were 'pure B', but only 5.8% of IM were. These two analyses showed that WM individuals truly belong to the wild taxon. Given the allelic frequencies of each morphotype, it is likely that a small proportion of individuals classed as WM were introgressed. Ideally, one would expect the same proportion of WM individuals to be classified as "pure B" as of allopatric individuals. Our results indicate that approximately 20% of individuals classed as WM may have in fact been introgressed. Nevertheless, in further analyses, we excluded individuals from the allopatric wild populations, to use only WM individuals as reference wild individuals. As members of the wild population actually involved in hybridisation, they should be a better reference for assessing hybridisation in Savane Manuel than are samples from allopatric wild populations. Furthermore, exclusion of the latter populations allowed us to make use of all six loci typed for WM, IM, JM and DM individuals. In any case, using as a reference WM individuals, including some that are likely introgressed, means that our estimations of the extent of hybridization are conservative This clearly proves they do not form a third entity, but rather are hybrids. Juveniles mostly had a mixed or "pure B" ancestry (Table 2, sub-run 2).

Factorial Correspondence Analysis

Figure 2 presents a projection of the individual coordinates on the principal plane of the FCA. DM, WM and IM morphotypes had very different means on the first factorial axis (P < 0.001 for all pairwise t-tests). IM individuals formed a continuum between the two other groups, consistent with their being hybrids.

Dynamics of this contact zone 43% of the 142 genotyped WM plants from Savane Manuel possessed only W alleles at each locus, and the remainder had at least one D allele. This is mostly due to the presence of three shared alleles with high frequencies, which were also present in the allopatric wild populations. This suggests that these alleles either represent shared ancestral polymorphism or are homoplasious. Of the 189 IM plants from Savane Manuel, 0.5% possessed only W alleles, 2.7% possessed, at each locus, one D allele and one W allele (as expected for F1 hybrids), 52.2% presented a multilocus genotype consistent with being post-F1 hybrids and the others had genotypes consistent with being backcrosses towards either wild or domesticated individuals. The remainder had genotypes consistent with backcrossing towards one of the parents (either domesticated or wild). Among the juveniles from Savane Manuel, 15.5% had only W alleles; 0.4% had the multilocus genotype expected for F1 hybrids, and 53.6% had a genotype expected for post-F1 hybrids.

The remainder had genotypes consistent with being backcrosses (mostly towards wild individuals).

Multilocus linkage disequilibrium (κ0,2) and Hardy-Weinberg disequilibrium (κ1,1) were then assessed using MULTILOCUS [START_REF] Barton | Estimating multilocus linkage disequilibria[END_REF], on the individuals from Savane Manuel. For the whole population as well as for the subsample of sexually mature plants, the chosen model was the most complex one, but for the JM sample, the model assuming equal δp among loci and no higher-order association coefficients was chosen.

Values of κ1,1 and κ0,2 are shown in Table 3.

In the entire population, κ1,1 was significant (κ1,1 = 4.94% [3.6-5.0%]) but far from its maximum possible value (13.6%, with the allelic frequencies present in DM and WM), suggesting the existence of a barrier to hybridisation (incomplete mixing of the populations), due either to genetic or reproductive incompatibility or to a spatial structure, or both. Spatial structure is the more likely explanation, as IM and WM individuals were not located randomly (see Fig. 1). This result was corroborated by the high, but also far from maximum, value of κ0,2 (4.13% [4.0-5.0%]), the estimator of multilocus linkage disequilibrium. If there is no admixture of the populations, κ0,2 should remain at its highest value (13.6%, given the allele frequencies found in Savane Manuel), whereas it should decrease by half at each generation if complete mixing occurs.

Our results show that at least the equivalent of 2-3 generations of free recombination must have occurred in Savane Manuel since the first hybridisation event.

Comparison of κ0,2 values in adult vs. juvenile subsamples (5.34% [4.9-7.0%] vs. 1.94% [1.5-2.4%]) suggests that linkage disequilibrium is currently decreasing, implying that recombination of wild and domesticated genomes is continuing, even if not necessarily completely unconstrained.

A classical representation of the admixture phenomenon is given in Figure 4, showing that most of the IM individuals were genetically intermediate between the two parental populations, and some were apparently strongly introgressed in one or the other direction, at these particular microsatellite loci.

Morphological differentiation between wild and domesticated populations

Six of the seven size variables (i.e. total height, height at first branching, leaf length and width, stem diameter, and leaf lobe number; stem number was the sole exception) decreased with hybrid index (t-tests, P < 0.001 for all tests). Most IM plants (62%) were larger than any wild-type plant studied.

Figure 5A shows the first factorial plane of the . Figure 5B shows the normalized distribution of PC1 of the different morphotypes. WM individuals were more homogeneous for PC1 than were DM individuals (Fisher test, P < 0.01), which were in turn less variable than IM individuals (Fisher test, P = 0.05). PC1 of the Hill & Smith analysis was also highly correlated with hybrid index (i.e., the frequency of the W synthetic allele within an individual, r² = 0.548, P < 0.001; Fig. 5C). Morphological variance depended on the hybrid index, being relatively large in intermediate and extreme D classes, and low in extreme W classes.

The geographic coordinate Y (position along the length of the transect) was also correlated with both PC1 of the Hill & Smith analysis (r² = 0.502, n = 291 adults; P < 0.001) and PC1 of the genetic FCA (r 2 = 0.435, n = 711 mature and juvenile plants; P < 0.001).

Identification of other small hybrid zones

Three small hybrid zones were identified about 100 km away from Savane Manuel (Kamuyene [04°58.2' N, 052°26.3' W]; a field near the southern limit of Macouria [04°58.2'N, 052°26.4' W] and Le Grand Blanc [04°58.8' N, 052°26.6' W]). Respectively, 2 out of 3, 2 out of 6 and 5 out of 12 individuals appeared to be hybrids (the others being wild; see the FCA analysis presented as Supplementary Material online). Furthermore, 71 individuals were sampled on the roadside from Savane Manuel to Sinnamary (3 km). In this environment, wild Manihot grows spontaneously (but is periodically mowed if close to the road) and some domesticated plants had been planted in nearby gardens or along the roadside. This was indeed a favourable context for hybridization between the two taxa, and 27 individuals (38%) were hybrids (4 were wild and 40 were domesticated).

DISCUSSION

Hybridisation within the genus Manihot Species boundaries in Manihot are "not sharply delimited" [START_REF] Rogers | Manihot, Manihotoides (Euphorbiaceae). Flora Neotropica[END_REF], and it has been argued that all taxa of the genus are interfertile [START_REF] Jennings | Cassava[END_REF]. Despite these claims, however, natural hybrids have rarely been documented, and never characterized on a molecular basis. Although [START_REF] Lefèvre | Isozyme diversity within African cassava germplasm[END_REF] noted that tree cassava (M. glaziovii) naturally hybridised with cassava in western Africa, they did not assess the fertility of the hybrids, and the identity of the wild taxon is unclear, as tree cassava is stated elsewhere to be itself an artificial hybrid between M. glaziovii and cassava [START_REF] Wanyera | Introgression of ceara rubber (Manihot glaziovii Muell-Arg) into cassava (Manihot esculenta Crantz): a morphological and electrophoretic evidence[END_REF]. Here, for the first time, we have demonstrated, based on molecular evidence, that a clonally propagated crop has hybridised in nature with a wild relative and that hybrids were fertile. Thus, selection associated with clonal propagation has not prevented cassava from retaining the capacity for sexual reproduction and from intercrossing with wild relatives. Detection of hybridisation in this site was greatly facilitated by the high degree of morphological and genetic differentiation presented by domesticated and wild populations in French Guiana. Indeed, cassava shares alleles (for both microsatellites and three nuclear genes) only with wild populations from the southern part of the Amazon basin, where domestication apparently took place [START_REF] Olsen | Evidence on the origin of cassava: Phylogeography of Manihot esculenta[END_REF][START_REF] Olsen | SNPs, SSRs and inferences on cassava's origin[END_REF]. In contrast, the wild Manihot taxon we studied in French Guiana is strongly differentiated from domesticated cassava, showing greater genetic affinity with populations of wild Manihot (M. pruinosa) along the southeastern rim of Amazonia (G. Léotard, unpublished data).

Frequency of hybridisation events

This study focused on only one hybrid zone, in which the hybrids possessed up to five alleles per locus private to WM populations and five exclusive to DM populations. This implies that at least three wild and three domesticated individuals were involved in the first hybridisation events. Moreover, the hybrids found between Savane Manuel and the town of Sinnamary possessed domesticated alleles absent from Savane Manuel. Their formation thus involved still other domesticated parents. Furthermore, three very small CROP-WILD NATURAL HYBRIDIZATION IN CASSAVA hybrid zones were identified about 100 km southeast of Savane Manuel. This suggests that the extensive hybridisation observed in Savane Manuel has also occurred in several other places. Hybridisation between cassava and this wild relative is thus not an isolated event in coastal French Guiana. It seems to occur easily whenever cassava is planted in proximity to wild populations in coastal savannas.

Heterosis and the genetic and morphological diversity of hybrids

Cassava is known to be a highly heterozygous crop [START_REF] Chavarriaga-Aguirre | Microsatellites in cassava (Manihot esculenta Crantz): discovery, inheritance and variability[END_REF], a characteristic allowed by clonal reproduction. In our sample, the average heterozygosity of domesticated individuals was 57%, similar to that found by [START_REF] Fregene | Simple sequence repeat marker diversity in cassava landraces: genetic diversity and differentiation in an asexually propagated crop[END_REF]. Both farmer selection (Pujol et al. 2005a) and natural selection [START_REF] Pujol | Size asymmetry in intraspecific competition and the density-dependence of inbreeding depression in a natural plant population: a case study in cassava (Manihot esculenta Crantz, Euphorbiaceae)[END_REF] help maintain a high rate of heterozygosity in planted clones. In contrast, wild individuals presented a substantially lower heterozygosity (41.7%). IM individuals, however, were even more heterozygous (61.8%) than were domesticated individuals, due to the mixing of D and W alleles. On the whole, genetic diversity was increased in hybrids, with no loss of rare alleles. In contrast to wild individuals, hybrids exhibited huge morphological variation (Fisher-Snedecor test, P < 0.01), consistent with their high genetic diversity and with the diversity of hybrid pedigrees (F1, F2, and probably backcrosses with the wild relative). Hybrids are expected to present high morphological variation, as they express a mosaic of parental phenotypes [START_REF] Allendorf | The problems with hybrids: setting conservation guidelines[END_REF]. Increased heterozygosity and genetic diversity in F1 hybrids -and to a lesser extent, in subsequent generations -is invoked in all cases of high hybrid vigour. Heterosis, the higher vigour of hybrids, has been observed in numerous situations, mainly involving plants (e.g. [START_REF] Arnold | Are natural hybrids fit or unfit relative to their parents?[END_REF], and can result from overdominance, masking of deleterious recessive alleles, increased mutation rate, and new allele combinations [START_REF] Jarvis | Wild relatives and crop cultivars: detecting natural introgression and farmer selection of new genetic combinations in agroecosystems[END_REF][START_REF] Rieseberg | Major ecological transitions in wild sunflowers facilitated by hybridisation[END_REF]. In this study, we did not evaluate the fitness of the hybrids. However, we did observe that hybrids, although likely younger, were much larger on average than were wild individuals. They can thus outcompete the wild parents in competition for light. Nevertheless, heterosis is often restricted to vegetative characters, and hybrid fitness is not always correlated with hybrid vigour; [START_REF] Dobzhansky | Nature and origin of heterosis[END_REF] described as "luxuriance" the case of interspecific hybrids that display some heterotic traits together with a reduced reproductive fitness. Therefore our observation of heterosis for size does not mean that Manihot hybrids are necessarily fitter than their wild parents. Moreover, we observed abnormal phenotypes, with a very low frequency of branching (and consequently of flowering) in eight hybrid individuals, genotypes of which were inconsistent with their being F1s, but compatible with F2 status. Low fitness often appears in the F2 as a consequence of the rupture of co-adapted gene complexes [START_REF] Barton | Analysis of hybrid zones[END_REF][START_REF] Rieseberg | Major ecological transitions in wild sunflowers facilitated by hybridisation[END_REF]. Another potential difference between hybrids and wild Manihot lies in ecological preferences. It has often been suggested that, although hybrids may have lower fitness than their parents in their respective niches, they may outcompete parental forms in some new ecological niche (the bounded hybrid superiority hypothesis [Arnold 1995 and references therein]). In the present study, even though hybrids belonged to several generations, they are clustered on a relatively small area of talus formed by the soil removed when the ditch was dug. Wild parents were found in a less disturbed part of the sampled area. Hybrids might be fitter than their wild parent only in well-drained disturbed micro-environments such as this talus. Importantly, the persistence of a strong spatial structure and ecological segregation of hybrids at a small scale, several generations after primary hybridisation, suggests either strong differential selection among environments or very low gene flow between the hybrid and wild compartments, or both. In our case, pollen is thought to be dispersed by stingless bees, and seeds are dispersed first balistically (through explosive fruit dehiscence), and then by ants. Seed dispersal in cassava thus probably occurs at a more local scale than pollen dispersal. The clustering of the hybrids suggests that pollen flow from the hybrid to the wild subpopulation is limited. Reduced male fertility in hybrids is a possible cause. Planting of wild and hybrid individuals in different controlled environments and assessment of their fertility would help in understanding genetic and environmental contributions to their relative vigour.

Fate of the hybrid zone: barriers to hybridisation, or gene swamping?

Despite some evidence for partial hybrid breakdown (see above), there seem to be no strong barriers to hybridisation in Savane Manuel. Hybrid index was highly variable (Fig. 4), with some intermediate individuals presenting an F1 pattern, but the great majority of them (96.7%) probably being post-F1 or backcross. Some F1s may be masked by the presence of a few shared alleles, but the very low value of κ0,2 (an estimator of linkage disequilibrium between alleles issuing from wild populations on the one hand and domesticated plants on the other) shows that hybridisation has in any case long since passed the F1 stage. Furthermore, dramatic reduction of κ0,2 in juveniles compared to adults shows that genetic mixing is still going on. One potential barrier is spatial (spatial clustering of the wild and hybrid gene pools), rather than biological. The currently restricted dispersal and gene flow should therefore not be considered a permanent feature of this hybrid zone. Ultimately, the fate of this hybrid zone will depend on the relative fitness of wild and hybrid individuals. If hybrids are as fit as wild individuals both on the talus and in the remainder of the savanna, domesticated genes may introgress into the wild population and the hybrid zone will become a uniform hybrid swarm. If, on the other hand, hybrids are fitter than wild individuals only on the better-drained talus, the swarm may remain confined there [START_REF] Allendorf | The problems with hybrids: setting conservation guidelines[END_REF]. However, in this very small contact zone, gene flow will oppose local adaptation. Hybrid zones can be stabilized in the long term only when selection is strong relative to gene flow, and affects many loci throughout the genome, or when there is a permanent input of parental forms from external populations [START_REF] Barton | Analysis of hybrid zones[END_REF]. Otherwise, any locus not tightly linked with a selected locus is expected to homogenize rapidly throughout the zone. This may well happen in Savane Manuel, given the small size and relative isolation of the contact zone, the apparent weakness of intrinsic barriers, and the evidence for effective recombination among markers (i.e., low linkage disequilibrium).

Conservation concerns

In Savane Manuel, gene flow was strongly asymmetrical, from domesticated to wild, probably because domesticated plants were removed some years ago. The mostly clonal propagation of domesticated cassava limits the incorporation of hybrids into the domesticated pool. Although Amerindian farmers regularly incorporate plants from self-sown seeds into their stocks of clones [START_REF] Elias | Unmanaged sexual reproduction and the dynamics of genetic diversity of a vegetatively propagated crop plant, cassava (Manihot esculenta Crantz), in a traditional farming system[END_REF], this practice seems to be much less frequent among the Creoles, Haitians and Surinamese who plant cassava in the coastal savannas of French Guiana (D. McKey, unpubl. observ.). Further analysis is needed to determine whether gene flow is asymmetric in all contact zones. Crop-to-wild gene flow could lead either to the appearance of weeds, as has been documented in bean (Papa & Gepts 2003) and beet (Viard et al. 2003), or to the introduction of domesticated genes into the wild genome, which could eventually lead to the extinction of wild populations by complete admixture [START_REF] Ellstrand | Gene flow and introgression from domesticated plants into their wild relatives[END_REF][START_REF] Allendorf | The problems with hybrids: setting conservation guidelines[END_REF]. This is most likely to happen when the wild populations are rather small [START_REF] Ellstrand | Gene flow and introgression from domesticated plants into their wild relatives[END_REF], as is often the case for wild Manihot species.

Gene flow from domesticated to wild taxa also raises the concern of transgene flow. Impacts of transgene flow are unclear, but could include decreased variability of wild taxa or cultivated landraces (Gepts & Papa 2003) or enhancement of the weediness of the feral taxon, as shown for several crops [START_REF] Ellstrand | Gene flow and introgression from domesticated plants into their wild relatives[END_REF]. The spread of a transgene will depend on its fitness effect. The fitness effect of a transgene depends both on the crop in which it is introduced and on the transgene itself, stressing the need for case-by-case studies (Den [START_REF] Nijs | Introgression from Genetically Modified Plants into Wild Relatives and its Consequences[END_REF]). Indeed, some transgenes impose fitness costs in the absence of the selective pressure (e.g., herbicide) for which they are designed, which would prevent their spread into non-target environments (e.g., [START_REF] Bartsch | Environmental implications of gene flow from sugar beet to wild beet -current status and future research needs[END_REF]. Others are neutral and are expected to diffuse neutrally in non-target environments (e.g., [START_REF] Burke | Fitness effects of transgenic disease resistance in sunflowers[END_REF], and others still can confer a selective advantage in a wide range of environments and are expected to spread widely into both domesticated plants and taxa intercrossing with them (e.g., [START_REF] Snow | A Bt transgene reduces herbivory and enhances fecundity in wild sunflowers[END_REF]. With the exception of potato [START_REF] Celis | Environmental biosafety and transgenic potato in a centre of diversity for this crop[END_REF], clonally propagated crop plants appear to have been neglected in discussions of biosafety concerns. Our study demonstrates the pertinence of this theme for cassava, on which development of transgenic plants is an active focus of current research [START_REF] Taylor | Development and application of transgenic technologies in cassava[END_REF][START_REF] Ihemere | Genetic modification of cassava for enhanced starch production[END_REF], and suggests the interest of conducting similar studies with other clonally propagated crops. Indeed, at least for cassava, selection for traits enhancing clonal propagation, which is in direct competition with sexual reproduction, has not led to reproductive isolation of the crop from wild relatives, and crop-wild hybridisation is neither an isolated phenomenon, nor an ephemeral process.

In the case of cassava, a hybrid bridge could allow gene flow to wild taxa not in direct contact with populations of the domesticated taxon. This bridge could be provided by a wild taxon such as the one we encountered in Savane Manuel. Hybrid zones such as those we have described need to be monitored over longer periods, in order to understand their dynamics and the evolutionary consequences of hybridisation.

Supplementary Figure 1: Factorial correspondence analysis on the matrix of allele counts per morphotype: projection of the individuals from four small hybrid zones, treated as supplementary individuals, on the principal plane. The analysis was conducted on the same individuals as those presented in Figure 2. The ellipses include 95 % of the individuals from each morphotype. Black squares: Kamuyene [04°58.2' N, 052°26.3' W]; grey diamonds: a field near the southern limit of Macouria [04°58.2'N, 052°26.4' W]; black triangles: Le Grand Blanc [04°58.8' N, 052°26.6' W]; white squares: individuals located between Savane Manuel and the town of Sinnamary, along the road.

-3 .0 -2 .5

Figure 1 :

 1 Figure 1: Localisation of the populations used in this study. A) Map of French Guiana, showing the sites where allopatric wild individuals were sampled (black circles). Number of individuals at each location appears between parentheses (note that sometimes populations are very close to each other, e.g., the three populations close to Kourou). Savane Manuel is indicated by the triangle; the 72 domesticated individuals used in the analysis were sampled in the closest fields (within 3 km). The other small hybrid zones are indicated by the black square. B) Map of the systematically sampled transect in Savane Manuel. Grey squares: IM individuals; black circles: WM individuals; dots: juveniles. DM individuals had been observed a few meters north (top of the map) of the sampled transect, but had been removed by the time of sampling.

Figure 2 :

 2 Figure 2: Factorial correspondence analysis on the matrix of allele counts per morphotype: projection of the individuals in the principal plane. White diamonds: DM individuals; grey squares: IM individuals; black circles: WM individuals; dots: juveniles.

Figure 4 :

 4 Figure 4 : Distribution of hybrid index per morphotype. DM individuals figure in white (69 individuals); IM in grey (184 individuals); WM in black (148 individuals) and juveniles in hatched grey (265 individuals).

Figure 5 :

 5 Figure 5: Hill and Smith analysis on ten morphological variables. A) Projection of the individuals on the principal plane. Individuals were grouped a posteriori according to their morphotype. B) Normalized distribution of PC1 of individuals from different morphotypes. Each curve represents the distribution of PC1 for one morphotype. Abbreviations: see Materials & Methods. Normalized distribution of PC1 of the same individuals, grouped by genotype composition in W allele (hybrid index HI). 1:0.000 ≤ HI ≤ 0.125; 2:0.125 < HI ≤ 0.250 … 8:0.875 ≤ HI ≤ 1.000.

Table 1 :

 1 Summary statistics averaged over 6 loci, for the four morphotypes. N: sample size; AR: number of alleles per locus; HO: observed heterozygosity; HE: expected heterozygosity (gene diversity); f:[START_REF] Weir | Estimating F-statistics for the analysis of population structure[END_REF] estimator of FIS, the intra-population fixation index; P-value associated with the exact test for deviations from Hardy-Weinberg proportions (computed using GENEPOP).

		N	AR	Ho	HE	f	P-value
	DM	72	5.91	0.570	0.671	0.152	< 0.001
	WM	148	5.71	0.417	0.561	0.253	< 0.001
	IM	184	6.12	0.618	0.713	0.134	< 0.001
	JM	265	6.11	0.453	0.681	0.335	< 0.001

Table 2 .

 2 Proportion of individuals having more than 90% ancestry in one cluster, in three different STRUCTURE runs. Run 1: K=2, no prior information, includes allopatric wild individuals (and thus only five loci). Run 2: K=2, no prior information, allopatric wild individuals not included. Run 3: K=3, prior population information for WM and DM individuals.

		Run 1		Run 2			Run 3
	Morphotype
		A	B	A	B	A	B	C
	DM	100.0%	0.0%	100.0%	0.0%	100.0%	0.0%	0.0%
	WM	0.7%	66.9%	0.7%	64.8%	0.7%	81.7%	0.0%
	Allopatric wild	2.0%	89.0%	--	--	--	--	--
	IM	38.6%	5.8%	21.7%	3.7%	0.0%	3.2%	7.4%
	JM	15.8%	33.%	11.3%	30.6%	0.0%	25.3%	3.8%
	RESULTS
	Intra-population genetic structure
	Allelic diversity was relatively high, with forty-seven
	alleles observed over the six loci for the plants sampled
	in Savane Manuel, and a total of fifty-five alleles when
	DM individuals were included. Table 1 summarizes
	statistics concerning genetic diversity across all six loci
	in the three morphotypes. Allelic richness was not
	significantly different among populations. Hardy-
	Weinberg expectations were not met, with a strong
	multilocus heterozygote deficit in all four
	morphological groups. More pairs of loci were in
	linkage disequilibrium among WM than in DM, IM or
	JM populations, probably due to a slight spatial
	structuring of the population (data not shown).

  CROP-WILD NATURAL HYBRIDIZATION IN CASSAVAmixture of the alleles present in DM and WM, mostly with intermediate frequencies. They did not display a subset of the alleles present in DM, ruling out the hypothesis that they could be escapees from cultivation.

	). Allopatric wild individuals exhibited
	allelic frequencies different from WM individuals, but
	the alleles encountered were the same, with the
	exceptions of allele 193 at locus GA126, being
	exclusively present in WM and IM individuals from
	Savane Manuel, and of allele 286 at locus SSR68,
	encountered only in allopatric wild populations. Some
	alleles (e.g., allele 140 at locus SSR55 and allele 246 at
	locus SSR68) are shared between DM and WM

populations, but were also present in allopatric populations. Several alleles were present in DM individuals, and in WM at low frequencies, but absent from allopatric wild samples (e.g., alleles 144 and 146 at locus GA12; see Figure

3

). IM individuals bore a

Table 3

 3

	κ0,2	κ1,1

.

2)

and inter-(κ1,1) genome associations, computed with MULTILOCUS MATHEMATICA add-ons

[START_REF] Barton | Estimating multilocus linkage disequilibria[END_REF]

. Maximum values of κ0,2 and κ1,1 are 13.6% in each case.

ACKNOWLEDGEMENTS

This study was funded by grants to D.

McKey from various agencies of the French government (programme 'Impact des Biotechnologies dans les Agroécosystèmes" of the Ministry of Research, programme "Ecosystèmes Tropicaux" of the Ministry of Ecology and Sustainable Development, the "Bureau des Ressources Génétiques", and the "Contrat Plan Etat-Région Guyane"). A. Duputié was supported by a stipend from the Ecole Normale Supérieure, Lyon. We thank the Office of the Mayor, Sinnamary, for permission to study in the site, as well as the farmers who allowed us to sample and measure the cassava varieties growing in their fields. Genotyping was done in the "Service Commun de Marqueurs Génétiques en Ecologie" of the CEFE. We thank G. Léotard, M. Delêtre and B. Pujol for permission to cite their unpublished results on G3pdh and microsatellite loci, and F. Massol for adapting the MULTILOCUS add-ons for 6 loci. We are grateful to J. D. Lebreton for invaluable comments on statistical analysis and for bringing to our attention the study by Hill and Smith.

AUTHOR INFORMATION BOX

This study is part of Anne Duputié's PhD thesis, a work aimed at understanding the ecology of the domestication of cassava, and the evolutionary relationships between cassava and its wild relatives. Patrice David is a population geneticist and evolutionary biologist working on various organisms and evolutionary issues. Chantal Debain manages the CEFE's molecular markers service. Doyle McKey is an evolutionary ecologist interested in plant-animal mutualism and plant domestication.


Guillaume Léotard
email: leotardmtp@yahoo.fr

Anne Duputié

Finn Kjellberg

Emmanuel J P Douzery

Chantal Debain

Jean-Jacques De Granville

Doyle Mckey

PHYLOGEOGRAPHY AND THE ORIGIN OF CASSAVA: NEW INSIGHTS FROM THE NORTHERN RIM OF THE AMAZONIAN BASIN

Keywords: domestication, G3pdh, Guianas, hybridization, introgression

The origin of cassava (Manihot esculenta Crantz) is still unclear, although several recent studies have addressed this issue [START_REF] Fregene | Variability of chloroplast DNA and nuclear ribosomal DNA in cassava (Manihot esculenta Crantz) and its wild relatives[END_REF][START_REF] Roa | AFLP analysis of relationships among cassava and other Manihot species[END_REF][START_REF] Olsen | Evidence on the origin of cassava: phylogeography of Manihot esculenta[END_REF][START_REF] Elias | Assessment of genetic variability in a traditional cassava (Manihot esculenta Crantz) farming system, using AFLP markers[END_REF][START_REF] Roa | Cross-species amplification of cassava (Manihot esculenta) (Euphorbiaceae) microsatellites: allelic polymorphism and degree of relationship[END_REF][START_REF] Olsen | Microsatellite variation in cassava (Manihot esculenta, Euphorbiaceae) and its wild relatives: further evidence for a southern Amazonian origin of domestication[END_REF][START_REF] Olsen | SNPs, SSRs and inferences on cassava's origin[END_REF]. [START_REF] Rogers | Manihot, Manihotoides (Euphorbiaceae). Flora Neotropica[END_REF] postulated that cassava was a "compilo-species", i.e. the result of hybridization events between several species, among them Manihot aesculifolia (Kunth) Pohl, a species endemic to Central America. On the other hand, Allem hypothesized the crop to be issued from a single species, Manihot esculenta, with two subspecies found only in the wild: Manihot esculenta ssp. flabellifolia (Pohl) Ciferri and Manihot esculenta ssp. peruviana (Muell. Arg.) Allem [START_REF] Allem | The origin of Manihot esculenta Crantz (Euphorbiaceae)[END_REF][START_REF] Allem | The primary gene pool of cassava (Manihot esculenta Crantz subspecies esculenta, Euphorbiaceae)[END_REF]. These wild taxa together have a broad ecological range, from southwestern Amazonia to the savannas of the Guianas. Molecular studies have favored the latter scenario, generally showing that cassava was domesticated only once, in South America, with no contribution from the Mesoamerican species pool, or at least not from M. aesculifolia [START_REF] Roa | AFLP analysis of relationships among cassava and other Manihot species[END_REF][START_REF] Olsen | Evidence on the origin of cassava: phylogeography of Manihot esculenta[END_REF][START_REF] Roa | Cross-species amplification of cassava (Manihot esculenta) (Euphorbiaceae) microsatellites: allelic polymorphism and degree of relationship[END_REF][START_REF] Olsen | Microsatellite variation in cassava (Manihot esculenta, Euphorbiaceae) and its wild relatives: further evidence for a southern Amazonian origin of domestication[END_REF][START_REF] Olsen | SNPs, SSRs and inferences on cassava's origin[END_REF]. Study of this and seven other Mesoamerican species (A. Duputié, unpublished data) show that all are only distantly related to cassava, excluding the possibility that cassava was domesticated in Central America. These studies, however, suffer several limitations. First, they have considered only a limited sample of domesticated cassava accessions. Second, they overlooked a part of the range of Manihot esculenta ssp. flabellifolia: while the taxon is distributed on an arc partially circling the Amazon basin, from eastern Bolivia westwards to central Brazil eastwards, and in the Guianas and eastern Venezuela northwards, these studies considered only samples from Brazil, thereby excluding any possibility of testing the hypothesis that cassava could have more than one center of domestication. Yet, several other crops have been shown to have been domesticated twice (e.g. the common bean [START_REF] Gepts | Phaseolin protein variability in wild forms and landraces of the common bean (Phaseolus vulgaris) -Evidence for multiple centers of domestication[END_REF]). The present study aims at filling some of these gaps. [START_REF] Olsen | Evidence on the origin of cassava: phylogeography of Manihot esculenta[END_REF] sampled accessions of M. esculenta ssp. flabellifolia and the closely related species M. pruinosa Pohl from Brazil and 20 accessions of cultivated cassava from the CIAT core collection, a collection constituted with the goal of representing cassava's genetic and morphological diversity worldwide [START_REF] Hershey | Definition of a core collection for cassava[END_REF]. We combined their sample of wild Manihot with samples of Manihot esculenta ssp. flabellifolia from the northern rim of the Amazonian basin, to cover most of the range of this taxon. Furthermore, to test the hypothesis that cassava could have more than one center of domestication, we also broadened the sample of domesticated cassava to include landraces cultivated in the Guianas, thus filling an acknowledged gap in earlier studies [START_REF] Olsen | Microsatellite variation in cassava (Manihot esculenta, Euphorbiaceae) and its wild relatives: further evidence for a southern Amazonian origin of domestication[END_REF]. Even though these samples all come from the same region, it has been shown that cassava genetic diversity in a single village in Guyana can be comparable to that in the CIAT (International Center for Tropical Agriculture) core collection [START_REF] Elias | Assessment of genetic variability in a traditional cassava (Manihot esculenta Crantz) farming system, using AFLP markers[END_REF]. Finally, we included samples of two additional wild species from South America and two outgroups. Specifically, we addressed the following questions: (i) Was cassava domesticated only once? (ii) Did post-domestication gene flow play a role in shaping the crop's diversity in French Guiana?

MATERIAL & METHODS

Plant material

Six wild Manihot species have been described in the Guianas [START_REF] Rogers | Manihot, Manihotoides (Euphorbiaceae). Flora Neotropica[END_REF][START_REF] Allem | A new species of Manihot (Euphorbiaceae) from the Brazilian Amazon[END_REF]. They include rainforest vines, and shrubs of savanna and savanna-forest ecotone. Within the latter, several taxa have been named, some of doubtful distinctness. One of them, M. surinamensis Rogers & Appan, was synonymized with M. esculenta ssp. flabellifolia by [START_REF] Allem | The origin of Manihot esculenta Crantz (Euphorbiaceae)[END_REF]. We sampled populations of this taxon (i) in coastal regions of French Guiana, where human activity is relatively high, and (ii) in rock-savanna islands on inselbergs located in the densely forested south of French Guiana, where recent gene flow with the domesticate or presence of escapees from cultivation are both highly unlikely. Sampling locations are presented in Figure 1 and detailed in Supplementary Table 1. One population (SM) was formerly in sympatry with domesticated cassava, which is no longer cultivated in the site. Natural hybridization has been shown to occur between the two taxa [START_REF] Duputié | Natural hybridization between a clonally propagated crop, cassava (Manihot esculenta Crantz) and a wild relative in French Guiana[END_REF], and we sampled individuals of domesticated, wild and intermediate phenotype (4, 4 and 3 individuals, respectively). Only the three individuals of wild phenotype which were found to be purely wild individuals in another study [START_REF] Duputié | Natural hybridization between a clonally propagated crop, cassava (Manihot esculenta Crantz) and a wild relative in French Guiana[END_REF] were included in this analysis. Individuals of domesticated or intermediate phenotype were analyzed as supplementary individuals. Our sample of domesticated cassava includes 37 plants (belonging to 31 landraces) from French Guiana and 49 plants (16 landraces and 33 seedlings) from the Amerindian village of Rewa, Guyana. Sampling details are given in Supplementary Table 2. Two individuals of Manihot glaziovii Muell. Arg. and three of M. aff. quinquepartita Huber ex Rogers & Appan were included, together with three individuals found in the Monts d'Arawa, initially determined as two different species (M. aff. quinquepartita and M. brachyloba Muell. Arg.). These species are more distantly related to cassava [START_REF] Chacón | Phylogenetic patterns in the genus Manihot (Euphorbiaceae) inferred from analyses of nuclear and chloroplast DNA regions[END_REF]. Two outgroup specimens were collected in French Guiana: Cnidoscolus urens (L.) Arthur, and Jatropha gossypiifolia L. [START_REF] Olsen | Evidence on the origin of cassava: phylogeography of Manihot esculenta[END_REF]

Figure 1: Populations of the wild relative of cassava sampled in the Guianas (hexagons), and populations of M. esculenta subsp. flabellifolia (squares) and of M. pruinosa (circles) studied by

. Shades of grey indicate elevational contourstopographic levels. Abbreviations: Brazilian states: Acre (AC), Goías (GO), Mato Grosso (MT), Rondônia (RO), Tocantins (TO); French Guiana (FG), Guyana (GU); populations in French Guiana: Grand Macoua (GM), Roche Blanche (RB), Trou Poissons (TP), Savane Manuelinnamary (SM), Kourou (KR), Tonate (TN), Camopi (CAM), Roche Touatou (RT), Monts d'Arawa (ARW), Petit Mitaraka (MIT). Filled symbols represent populations containing G3pdh haplotypes shared with domesticated cassava.

DNA sequences

We sequenced a 962-bp long portion of the nuclear gene G3pdh, encompassing four exons and three introns, one of which contains a minisatellite region, as described by [START_REF] Olsen | Evidence on the origin of cassava: phylogeography of Manihot esculenta[END_REF]. The sequences of the 28 G3pdh haplotypes already identified by [START_REF] Olsen | Evidence on the origin of cassava: phylogeography of Manihot esculenta[END_REF] were obtained from GenBank (accession numbers AF136119-AF136149). DNA was extracted from dried leaves using DNeasy Plant kit (Qiagen GmbH). PCR amplification of the G3pdh region was performed as described in [START_REF] Olsen | Evidence on the origin of cassava: phylogeography of Manihot esculenta[END_REF] and sequencing reactions were performed using classical protocols with products from Applied Biosystems, on an ABI 310 monocapillary sequencer. We sequenced the two haplotypes of heterozygotes at the G3pdh locus together, using the "haplotype subtraction" approach [START_REF] Clark | Inference of haplotypes from PCR-amplified samples of diploid populations[END_REF], as did [START_REF] Olsen | Evidence on the origin of cassava: phylogeography of Manihot esculenta[END_REF]. Alignment was done using CLUSTAL X [START_REF] Thompson | The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools[END_REF] and manually corrected using BioEdit [START_REF] Hall | BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT[END_REF].

Phylogenetic analysis and analysis of molecular diversity

The sequenced region contained 89 variable sites within the genus Manihot. Translation of the exon sequences revealed a single amino acid substitution. One phylogenetically informative character, a substitution in position 309, was not used in the analysis, due to amplification ambiguities. The indel corresponding to the minisatellite region, comprising up to five repeats of a 25-bp motif, has already been shown to present strong homoplasy [START_REF] Olsen | Minisatellite variation in a single-copy nuclear gene: phylogenetic assessment of repeat length homoplasy and mutational mechanism[END_REF], and was therefore not taken into account when defining haplotypes, nor in drawing the haplotype network. The haplotype network was obtained by manual addition of the new sequences to the network presented in [START_REF] Olsen | Evidence on the origin of cassava: phylogeography of Manihot esculenta[END_REF]. Phylogenetic reconstruction using maximum likelihood was conducted using the program PHYML version 2.4.4 [START_REF] Guindon | A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood[END_REF], using the GTR + Γ + I substitution model. Indels were coded as missing characters. Estimates of within-population diversity (genetic diversity and nucleotide diversity [START_REF] Nei | Molecular Evolutionary Genetics[END_REF]) were calculated using the program ARLEQUIN [START_REF] Excoffier | Arlequin (version 3.0): An integrated software package for population genetics data analysis[END_REF].

RESULTS

We identified 12 new haplotypes in the genus Manihot (accession numbers: AM901263-AM901274): three in domesticated cassava (haplotypes D1 to D3), four in Manihot esculenta ssp. flabellifolia (haplotypes G1 to G4) two in M. glaziovii (GL1 and GL2) and three in M. aff. quinquepartita (BA1 to BA3). Forty-two sites were phylogenetically informative.

Monophyly of the genus Manihot is strongly supported by a bootstrap value of 100 %, but G3pdh sequences do not sort species within the genus Manihot (Supplementary Figure 1). The haplotype network (modified from [START_REF] Olsen | Evidence on the origin of cassava: phylogeography of Manihot esculenta[END_REF]) is shown in Figure 2.

M. esculenta ssp. flabellifolia populations from French Guiana Only four haplotypes, all of them never previously documented, were encountered in the 46 accessions from French Guiana. These haplotypes differed by at most four substitutions from alleles already documented in Manihot esculenta ssp. flabellifolia (Figure 2). Allele counts are given in Table 1. Strong differences in haplotype frequencies between populations of coastal savannas (G1: 82 %, G2: 11 %, G3: 5 %) and those of rock-savannas on inland inselbergs (G1: 37 %, G2: 35 %, G3: 28 %) suggest geographic structuring. Genetic diversity in Manihot esculenta ssp. flabellifolia from coastal populations was lower than in inland sites, this diversity being in turn lower than that found in Brazilian populations of the taxon (except those from central Brazil; Table 1). Nucleotide diversity was much lower among French Guianan populations than among Brazilian accessions, as the four Guianan haplotypes differed from each other by at most seven substitutions, as compared to up to 24 substitutions between Brazilian haplotypes.

Domesticated cassava

We detected eight haplotypes of G3pdh in 86 cassava accessions from the Guianas. Combined with earlier data [START_REF] Olsen | Evidence on the origin of cassava: phylogeography of Manihot esculenta[END_REF], ten G3pdh haplotypes have been documented so far in cassava, of which six are shared with populations of M. esculenta ssp. flabellifolia from the southern Amazonian rim (from Mato Grosso to Acre), and one is shared both with these populations and with populations of M. pruinosa from Goiás (Table 1, Figure 1). All three new cassava G3pdh haplotypes documented in this study differ from already documented haplotypes by only one mutation. The two haplotypes that dominated in [START_REF] Olsen | Evidence on the origin of cassava: phylogeography of Manihot esculenta[END_REF] sample also predominated in ours: haplotypes A and P had a cumulative frequency of 88 % in Guianan landraces, as compared to 85 % in Olsen and Schaal's sample from the CIAT world core collection (Table 1). Although we scored more alleles than did Olsen and Schaal, gene diversity and nucleotide diversity were much lower in our sample than in the 20 accessions of diverse origins included in their study (Table 2). [START_REF] Olsen | Evidence on the origin of cassava: phylogeography of Manihot esculenta[END_REF]. Haplotype names are as in Table 1, and the shapes around them indicate the taxa in which the haplotypes were present (diamonds: domesticated cassava; squares: M. esculenta ssp. flabellifolia from Brazil; hexagons: M. esculenta ssp. flabellifolia from French Guiana; circles: M. pruinosa; spiny circles: more distant species of Manihot). Each dash represents a single mutational step. The numbers on the lines indicate the position of the substitution with reference to the alignment; i and d followed by numbers indicate an insertion or deletion and its length. For branches with more than one mutational step, the order of the mutations is arbitrary. Multiple substitution events are followed by letters a and b. Homoplasic mutations are indicated by H.

Interspecific relationships

In Savane Manuel, the individuals of domesticated phenotype were all homozygous for haplotype A, while three of the four individuals of wild phenotype were homozygous for G1 or G2. The fourth one and all three individuals of intermediate phenotype were heterozygous, with one copy of haplotype A and one of either G1, G2 or G3, consistent with their being hybrids or introgressed individuals. The two samples of M. glaziovii were heterozygous, with one copy of haplotype P and one of either GL1 (individual from French Guiana) or GL2 (individual from Indonesia).

Finally, the three individuals from the Monts d'Arawa, initially determined as two different species, were all homozygous for haplotype BA1. Two other haplotypes (BA2 and BA3) were detected in M. aff. quinquepartita from other localities. DISCUSSION

Site of domestication

Our sample, combined with [START_REF] Olsen | Evidence on the origin of cassava: phylogeography of Manihot esculenta[END_REF], now covers most of the range of Manihot esculenta ssp. flabellifolia, as defined by [START_REF] Allem | The origin of Manihot esculenta Crantz (Euphorbiaceae)[END_REF]. Our data do not support a second center of domestication for cassava on the northern rim of the Amazonian basin, and reinforce [START_REF] Olsen | Evidence on the origin of cassava: phylogeography of Manihot esculenta[END_REF] conclusions, i.e., that cassava was domesticated only once, on the south-western Amazonian rim. In addition to haplotypes A, H, N, P, δ and ε already observed by [START_REF] Olsen | Evidence on the origin of cassava: phylogeography of Manihot esculenta[END_REF] in domesticated cassava, we found another haplotype (M) shared between Guianan cassava accessions and a population of M. esculenta ssp. flabellifolia from Jaru (Rondônia, south-western Amazonian rim), and three new haplotypes (D1, D2, D3) directly derived from haplotypes A and α of [START_REF] Olsen | Evidence on the origin of cassava: phylogeography of Manihot esculenta[END_REF] Figure 2), further supporting a single domestication event for the crop.

Genetic diversity in cassava and its closest wild relatives

The same alleles predominated in our samples of cultivated cassava and those of [START_REF] Olsen | Evidence on the origin of cassava: phylogeography of Manihot esculenta[END_REF] In another case, hybridization seems clearly the more likely explanation for patterns observed. Both M. glaziovii individuals studied bore one copy of haplotype P, frequent in cassava but very different from the other haplotype borne by these individuals (GL1 and GL2, differing by only six substitutions). Manihot glaziovii has been widely introduced by humans, as a source of latex (Para rubber) and as a shade tree, and has been subjected to crossing with domesticated cassava in programs aimed at increasing disease resistance and productivity of cassava [START_REF] Jennings | Cassava[END_REF]. Does hybridization occur between more closely related species, and can it be detected using this marker? As already stated by [START_REF] Olsen | Evidence on the origin of cassava: phylogeography of Manihot esculenta[END_REF], there is little evidence of hybridization between Manihot pruinosa and Manihot esculenta, because only two alleles (out of 70: haplotypes E and J, each found once) scored in this taxon were shared with a sympatric species (M. esculenta ssp. flabellifolia). Within the M. esculenta group, hybridization is possible, as shown in Savane Manuel [START_REF] Duputié | Natural hybridization between a clonally propagated crop, cassava (Manihot esculenta Crantz) and a wild relative in French Guiana[END_REF]. However, no haplotype of M. esculenta ssp.

flabellifolia from the Guianas was found in cultivated cassava there, suggesting that hybridization between these two taxa is only a recent and local event. In French Guiana, cassava is traditionally cultivated in forest openings, under slash-and-burn systems, while its wild relatives are savanna shrubs. Only recently has cassava begun to be marginally cultivated in small savanna areas in this region. We may surmise that ongoing deforestation in other regions where cassava and its wild relatives co-occur may create similar situations of secondary contact, hybridization and introgression. Further insight could be gained from studies in areas of contact between wild and domesticated populations where Amerindian farmers incorporate volunteer seedlings and hence individuals of possibly hybrid origin [START_REF] Elias | Assessment of genetic variability in a traditional cassava (Manihot esculenta Crantz) farming system, using AFLP markers[END_REF].

Table 1 .

 1 Distribution of G3pdh haplotypes in the studied populations of M. esculenta ssp. flabellifolia, M. pruinosa and domesticated cassava. Haplotypes in light grey correspond to haplotypes found in domesticated cassava, and haplotypes in darker grey correspond to those found in wild Manihot in the Guianas. n represents the number of haplotypes (i.e., twice the number of sample

	Population	n

Brazilian M. esculenta ssp. flabellifolia (Olsen & Schaal, 1999)

	Rondônia and Acre	10		7		5 12	3	4	17 3 21 4	6 4
	Mato Grosso 8 3		11			1	10 15	2	6	6 5	1
	Goiás		48 1				47
	Tocantins		4 25	6 1			3	11 2
	M. pruinosa (Olsen & Schaal, 1999)
	Mato Grosso	9 12			2	1
	Goiás	9	1	1	18	17
	Cultivated cassava
	Local landraces [FG; GU] (this study) 172					1	5 2 5	6	1 140 12
	World Core Collection (Olsen & Schaal, 1999)	1					1 2		19 15 2
	Domesticated cassava, total 212	1				1	6 4 5	6	1 159 27 2

Table 2 .

 2 Gene diversity and nucleotide diversity of Manihot esculenta ssp. esculenta, M. esculenta ssp. flabellifolia and M. pruinosa at the regional level.

		Sample size	Gene diversity (± s.d.)	Nucleotide diversity (x10 3 ) (± s.d.)
	Manihot esculenta ssp. esculenta
	(cultivated cassava)
	Local landraces (FG and GU)	86	0.331 +/-0.046	1.5 +/-1.0
	World core collection (data from Olsen and Schaal, 1999)	20	0.644 +/-0.049	5.5 +/-3.1
	Manihot esculenta ssp. flabellifolia
	Acre-Rondônia	48	0.884 +/-0.015	14.5 +/-7.3
	Mato Grosso	35	0.884 +/-0.015	16.3 +/-8.2
	Goías	48	0.516 +/-0.013	19.6 +/-9.7
	Tocantins	26	0.713 +/-0.051	20.0 +/-10.0
	French Guiana -inland	19	0.679 +/-0.023	2.8 +/-1.7
	French Guiana -coast	27	0.297 +/-0.076	1.4 +/-1.0
	Manihot pruinosa
	Mato Grosso	12	0.627 +/-0.062	13.2 +/-6.9
	Tocantins	23	0.686 +/-0.032	14.8 +/-7.5

Figure 2: G3pdh haplotype network (modified from

  M. brachyloba with trilobate leaves and an undetermined species with deeply dissected leaf lobes) in the Monts d'Arawa were fixed for a single G3pdh haplotype (BA1). Subsequent field work showed that leaf morphology of M. aff. quinquepartita varies over the life cycle in a predictable way (D. McKey, unpublished data) and the re-examination of the three voucher specimens after field work suggested that all three individuals were indeed M. aff. quinquepartita. Do gene sequences enable us to sort species? G3pdh sequences showed that the genus Manihot was monophyletic with regard to Jatropha and Cnidoscolus, but did not sort species. Some species (e.g., cassava),

	Species boundaries, interspecific hybridization and
	Manihot taxonomy
	Species delimitations are far from clear in Manihot.
	Distinctions on purely morphological grounds can lead
	to mistakes, as some traits are highly variable within
	species, owing either to genetically based variation or
	to environmental or developmental plasticity. For
	example, two leaf morphs are present in populations of
	Guianan M. esculenta ssp. flabellifolia. The form with
	narrow lobes was distinguished by Rogers and Appan
	(1973) as M. surinamensis, but neither G3pdh nor
	microsatellite data (unpublished data) suggest any
	genetic differentiation between them. Similarly, what
	first appeared, from herbarium vouchers, to be three
	separate species (M. aff. quinquepartita with five-lobed
	leaves, show astonishing genetic diversity. Some cassava
	haplotypes (e.g., haplotypes M and P) are also shared
	with closely related species, suggesting either shared
	ancestral polymorphism -gene phylogenies are not
	always the same as species phylogenies -or
	hybridization.
	, showing that traditional landraces from the
	Guianas share the same genetic basis as do cultivars
	from other locations.
	Haplotype diversity of Manihot esculenta ssp.
	flabellifolia is geographically structured, and is highest
	in southwestern Amazonia, decreasing towards the
	east and north. This may indicate a population
	expansion from southwestern Amazonia eastwards and
	northwards, through a dry corridor that may have
	existed repeatedly during glacial times (van der
	Hammen and Hooghiemstra, 2000). The weak gene
	diversity and low differentiation in Guiana in M.
	esculenta ssp. flabellifolia suggest a strong bottleneck
	effect followed by limited subsequent differentiation
	by nucleotide substitution.

ACKNOWLEDGMENTS

We are grateful to François Massol for critical reading of the manuscript, and to an anonymous reviewer who helped us improve a previous version. Thanks to Marianne Elias, Marie Fleury, Philippe Gaucher, Clément Lermyte, Sandrine Manusset, Benoît Pujol and Corinne Sarthou for providing some of their samples. This study was funded by grants to D. McKey from various agencies of the French government (programme 'Impact des Biotechnologies dans les Agroécosystèmes' of the Ministry of Research, programme 'Écosystèmes Tropicaux' of the Ministry of Ecology and Sustainable Development, Bureau des Ressources Génétiques and Contrat Plan État Région Guyane). G. Léotard and A. Duputié were supported by doctoral thesis grants from the French Ministry of Research. Genotyping was done in the "Service Commun des Marqueurs Moléculaires en Écologie" of the CEFE. The contribution of E.J.P.D. is N° 2009-037 of the Institut des Sciences de l'Évolution de Montpellier (UMR 5554 -CNRS).

Supplementary Information for

Phylogeography and the origin of cassava: new insights from the northern rim of the Amazonian basin Sampling locations for the wild relatives of cassava. 1. Sampling locations for the wild relatives of cassava.

Supplementary Table

Species

Site

GPS coordinates

Number of samples

Name of collector

Cultivators