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

Marc Delêtre

Jean-Jacques De Granville

Doyle Mckey

POPULATION GENETICS OF MANIHOT ESCULENTA SSP. FLABELLIFOLIA GIVES INSIGHT INTO PAST DISTRIBUTION OF XERIC VEGETATION IN A POSTULATED FOREST REFUGIUM AREA IN NORTHERN AMAZONIA

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Introduction

Climate variation due to Milankovitch oscillations has triggered repeated shifts in species distributions [START_REF] Hewitt | The genetic legacy of the Quaternary ice ages[END_REF]. During glacial periods, climate in the tropics was cooler and drier than now, whereas it was warmer and wetter during interglacials [START_REF] Hewitt | The genetic legacy of the Quaternary ice ages[END_REF]. Several theories explaining patterns of species distribution in Amazonia have been proposed. On the one hand, the long-debated refugia theory proposed by [START_REF] Haffer | Speciation in Amazonian forest birds[END_REF] postulates that forests have expanded during interglacials, and receded during glacial times, becoming restricted to small islands (refugia) in a matrix of savanna vegetation or dry forest [START_REF] Pennington | Neotropical seasonally dry forests and Quaternary vegetation changes[END_REF]. Allopatric speciation of forest species would have been favoured by such stable forest refugia, and the regions of high forest species endemism, such as the Guianas [START_REF] Haffer | Speciation in Amazonian forest birds[END_REF][START_REF] De Granville | Rain forest and xeric flora refuges in French Guiana[END_REF][START_REF] De Granville | L'endémisme en Guyane française[END_REF][START_REF] Bush | Amazonian speciation: a necessarily complex model[END_REF], would have been forest refugia. On the other hand, [START_REF] Bush | Amazonian speciation: a necessarily complex model[END_REF] disturbance-vicariance hypothesis proposes that high endemism in the Guianas is due not to long-term stability of this region, but to numerous climate changes in this region. High endemism would be due to high rates of speciation, favoured by selection pressures due to environmental change. According to this view, vegetation adapted to drier climates covered much of the Guianas during glacial times, with rainforest expanding during interglacials.

Data supporting one or the other hypothesis in the Guianas are scarce. We are aware of only one site in inner French Guiana where palynological studies have been conducted, in the central part of French Guiana [START_REF] Charles-Dominique | Forest perturbations and biodiversity during the last ten thousand years in French Guiana[END_REF][START_REF] Ledru | Late Holocene rainforest disturbance in French Guiana[END_REF]. This site remained forested for the last 10,000 years [START_REF] Charles-Dominique | Forest perturbations and biodiversity during the last ten thousand years in French Guiana[END_REF], although primary forest was temporarily replaced by secondary forest at least twice, around 1,500 BP and around 1,000 BP [START_REF] Ledru | Late Holocene rainforest disturbance in French Guiana[END_REF]. Results of charcoal analyses conducted on soil cores [START_REF] Tardy | Paléoincendies naturels, feux anthropiques et environnements forestiers de Guyane Française du Tardiglaciaire à l'Holocène récent -Approches chronologique et anthracologique[END_REF], together with those arising from population genetic studies of a rainforest tree species, Vouacapoua americana [START_REF] Dutech | Tracking a genetic signal of extinction-recolonization events in a neotropical tree species: Vouacapoua americana Aublet in French Guiana[END_REF][START_REF] Dutech | 's website. and genetic diversity within French Guianan populations of a rainforest tree species, Vouacapoua americana[END_REF], were consistent with this central region being a forest refugium since the Last Glacial Maximum (LGM). However, no studies so far have considered the remote southern part of French Guiana, which is characterized by a drier forest type than in the remainder of the country (Gond et al. 2005). Vegetation models for the LGM suggest that the central part of French Guiana, as well as the western coastal region, were covered by rainforest vegetation during the LGM, whereas southern and eastern regions harboured somewhat drier vegetation [either dry forest or savannas; [START_REF] Bush | Amazonian speciation: a necessarily complex model[END_REF]; van der [START_REF] Van Der Hammen | Neogene and Quaternary history of vegetation, climate, and plant diversity in Amazonia[END_REF]; [START_REF] Pennington | Neotropical Savannas & Dry Forests: Plant diversity, Biogeography and Conservation[END_REF]]. However, climate simulations by [START_REF] Mayle | Responses of Amazonian ecosystems to climatic and atmospheric carbon dioxide changes since the last glacial maximum[END_REF] infer the continuous presence of evergreen broad-leaved forest in the whole region ever since the last glacial maximum.

Phylogeogeographic studies may provide insights into past vegetation. To cast light on the history of the vegetation of the Guianas, we examined the population genetics of a plant restricted to dry environments, at the scale of French Guiana. The species we considered, Manihot esculenta ssp. flabellifolia (Pohl) Ciferri, is the wild progenitor of cassava [START_REF] Olsen | Evidence on the origin of cassava: Phylogeography of Manihot esculenta[END_REF], with which it has been shown to occasionally hybridize in nature, at least in the coastal savannas of French Guiana [START_REF] Duputié | Natural hybridization between a clonally propagated crop, cassava (Manihot esculenta Crantz) and a wild relative in French Guiana[END_REF]. Manihot esculenta ssp. flabellifolia is distributed on an arc partly encircling the Amazon basin, from eastern Bolivia and Peru eastward to northeastern Brazil, northward to the Guianas and then westward to Venezuela. In the southwestern part of its distribution range, the plant grows in transition zones between forest and cerrado and can persist as a climbing shrub growing in openings in the forest [START_REF] Allem | The origin of Manihot esculenta Crantz (Euphorbiaceae)[END_REF][START_REF] Olsen | Evidence on the origin of cassava: Phylogeography of Manihot esculenta[END_REF], but it also grows as a bushy shrub in open areas in this region (A. Duputié, D. McKey, pers. obs.). In the northeastern part of its distribution range, the taxon is found in open environments such as savannas and granitic outcrops. Its distribution range in the Guianas is very patchy, as it is found in a narrow stretch along the coast, and forms small populations on rocky outcrops (inselbergs) far away from the coast, a distribution pattern it shares with numerous other species restricted to dry environments (de Granville 1982). In inner French Guiana, where forest openings and river banks could offer suitable habitats, Manihot esculenta ssp. flabellifolia has never been found. It is also absent from inselbergs located in northeastern French Guiana (Figure 1).

In contrast to cassava, which farmers propagate clonally using stem cuttings, vegetative reproduction appears to play no role in the biology of Manihot esculenta ssp. flabellifolia. Observations suggest that the taxon is pollinated mostly by meliponine bees. Seeds are dispersed first by ballistic autochory, through explosive dehiscence of the fruit, and then are secondarily dispersed by ants (notably Ectatomma brunneum and Dorymyrmex pyramicus ssp. guyanensis in the Guianas [START_REF] Elias | The unmanaged reproductive ecology of domesticated plants in traditional agroecosystems: an example involving cassava and a call for data[END_REF]). Seeds germinate in response to disturbance and/or opening of the environment [START_REF] Pujol | Germination ecology of cassava (Manihot esculenta Crantz, Euphorbiaceae) in traditional agroecosystems: seed and seedling biology of a vegetatively propagated domesticated plant[END_REF]. As meliponine bees have a flight range of only 0.1 -1 km [START_REF] Roubik | Ecology and Natural History of Tropical Bees[END_REF], and as seed dispersal does not exceed a few meters [START_REF] Elias | Sélection naturelle, sélection humaine, et diversité chez une plante domestiquée propagée de façon végétative: le cas de la culture du manioc par les Indiens Makushi du Guyana[END_REF], most dispersal events occur over a very short range. Because the germination of Manihot esculenta ssp. flabellifolia is conditioned to canopy opening, and as its closest relatives are restricted to open environments (Duputié et al., unpubl. data), one can hypothesize that this taxon has been restricted to open environments over the past few thousand years.

Genetic characteristics of Manihot populations from coastal and inner French Guiana may help cast light on the history of This is a postprint version of a paper published in Molecular Ecology (2009) 18:2897-2907; doi: 10.1111/j.1365-294X.2009.04231.x and available on the publisher's website.

vegetation in this part of Amazonia. If the northern part of the region (excluding the coastal savanna corridor) has remained forested during the past few thousand years, as postulated by [START_REF] Haffer | Speciation in Amazonian forest birds[END_REF][START_REF] De Granville | Rain forest and xeric flora refuges in French Guiana[END_REF] and [START_REF] Dutech | 's website. and genetic diversity within French Guianan populations of a rainforest tree species, Vouacapoua americana[END_REF], one should expect this zone to have formed a barrier to the dispersal of Manihot esculenta ssp. flabellifolia. Populations from the coastal savannas should therefore be strongly differentiated from populations of inner savannas. If the southern part of the region also remained forested, as [START_REF] Haffer | Speciation in Amazonian forest birds[END_REF] postulated, species restricted to dry environments should have been historically restricted to small xeric islands (inselbergs). Under this scenario, populations of M. esculenta ssp. flabellifolia in the southern part of the region would have been founded through occasional long-distance colonization of a few migrants. Hence, a strong isolation by distance should be expected for the inselberg populations of southern French Guiana. These populations would be expected to be strongly differentiated from one another and to have low intra-population diversity, due to genetic drift and to founder effects. On the other hand, if the southern part of the Guianas was formerly covered by drier vegetation types, a hypothesis defended by [START_REF] Bush | Amazonian speciation: a necessarily complex model[END_REF], inselberg populations of Manihot would be the remnants of formerly more extensive populations. Inter-population differentiation would be due to drift and mutation acting independently in each population, eventually obscuring underlying signals of isolation by distance. Intra-population diversity could have remained high.

Here, we (1) assess whether genetic structure of Manihot populations provides any evidence for past continuous dry vegetation areas in French Guiana (particularly in the southern region), and (2) determine the relationships between inselberg and coastal populations of Manihot in this region. To that aim, it is first necessary to assess whether, and to what extent, gene flow from domesticated cassava has affected the genome of its wild relative in French Guiana.

Material & Methods

Sampling

Leaf samples were picked from 373 individuals of Manihot esculenta ssp. flabellifolia (sensu [START_REF] Allem | The origin of Manihot esculenta Crantz (Euphorbiaceae)[END_REF]) from all over French Guiana, between 2002 and 2007. Ten populations were sampled along the coast and four on inselbergs (filled squares on Figure 1; sampling locations are presented in greater detail in Supplementary Table 1). One of these, SM, is the population where hybrids with cassava had been found by [START_REF] Duputié | Natural hybridization between a clonally propagated crop, cassava (Manihot esculenta Crantz) and a wild relative in French Guiana[END_REF]. Only purely wild individuals from this population were included. On the coast, we found no Manihot populations west of Savane Grand Macoua nor east of Cayenne. As inland inselbergs are only easily accessible by helicopter, we were only able to sample a limited number. The taxon probably can be found on other inselbergs we were not able to visit (dots on Figure 1). On the other hand, it was not present on all inselbergs we visited in the southern region, and has never been found on inselbergs in the northeastern region (open squares on Figure 1). The map showing the occurrence of Manihot on inland inselbergs was constructed using all samples present at the herbarium of Cayenne (CAY) and based on the compilation of unpublished notes and field observations of researchers who have visited these inselbergs. This is a postprint version of a paper published in Molecular Ecology (2009) 18:2897-2907; doi: 10.1111/j.1365-294X.2009.04231.x and available on the publisher's website.

In each population, 5 to 60 individuals were sampled randomly. Our sample size for each population is not representative of the actual population size: coastal populations are very large, whereas inselberg populations are of several tens to a few hundred individuals. Four very close inselbergs were sampled for population MA, three for population WA, and two for population RT (Figure 1). Even though our sampling design is not balanced, removal of the four populations with < 19 individuals did not modify our conclusions (Supplementary Material).

Genotyping

DNA was extracted using DNeasy 96 Plant kit (QIAGEN GmbH). On each extraction plate, six wells were dedicated to data control (blank and replicates). We scored seven microsatellite loci: GA12, GA21, GA57, GA126 (Chavarriaga-Aguirre et al. 1998), SSR55, SSR68 and SSR169 [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 loci were amplified jointly, using the multiplex PCR Taq from Qiagen, following the manufacturer's recommendations for a final volume of 10 µL. Amplifications were conducted on PTC-100 thermocyclers (MJ research) and genotyping was performed on an ABI 3130 sequencer. Genotypes were then eye-checked under GENEMAPPER 3.0 software (Applied Biosystems). Control blank wells never showed peaks, and no mismatch between the replicates was detected. Typing error rate was therefore low.

Data analysis

Before performing any analysis, we first looked for individuals introgressed by domesticated cassava. [START_REF] Duputié | Natural hybridization between a clonally propagated crop, cassava (Manihot esculenta Crantz) and a wild relative in French Guiana[END_REF] showed that, although the microsatellites we used were designed for domesticated cassava, they amplify readily in the wild taxon of French Guianan savannas, with alleles different from those found in domesticated cassava. Therefore, we sought individuals bearing alleles characteristic of domesticated individuals. The individuals used as a reference for domesticated alleles were a combination of the cultivated cassava clones used in [START_REF] Duputié | Natural hybridization between a clonally propagated crop, cassava (Manihot esculenta Crantz) and a wild relative in French Guiana[END_REF], and of over 400 cassava plants collected in different locations all over French Guiana (unpublished data).

Allelic richness AR (computed using the rarefaction method as suggested by El Mousadik & Petit [1996], and averaged over all loci), Weir & Cockerham's F-statistics (f, estimator of FIS, and θ, estimator of FST; [START_REF] Weir | Estimating F-statistics for the analysis of population structure[END_REF], and Nei's gene diversity H (expected heterozygosity, Nei 1987) were computed with FSTAT [START_REF] Goudet | FSTAT (Version 1.2): A computer program to calculate Fstatistics[END_REF]. P-values for f were assessed using the exact test implemented in GENEPOP [START_REF] Raymond | GENEPOP (Version 1.2) -Population genetics software for exact tests and ecumenicism[END_REF] with default settings. Comparisons of allelic richness and gene diversities were conducted in FSTAT using 10,000 permutations of genotypes among populations. Population differentiation tests were conducted in FSTAT, by permuting genotypes among populations 5,000 times. As multiple tests were performed, significance of the P-values was assessed using [START_REF] Benjamini | On the adaptive control of the false discovery rate in multiple testing with independent statistics[END_REF] sharpened test, as implemented in [START_REF] Verhoeven | Implementing false discovery rate control: increasing your power[END_REF]. Isolation by distance was assessed according to [START_REF] Rousset | Genetic differentiation and estimation of gene flow from Fstatistics under isolation by distance[END_REF] in GENEPOP.

Genetic structure of the populations was further explored using the Bayesian clustering algorithm implemented in STRUCTURE v.2.2 [START_REF] Pritchard | Inference of population structure using multilocus genotype data[END_REF]. The optimal number of clusters was assessed using the method of [START_REF] Evanno | Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study[END_REF], running the program ten times for each value of the allowed number of clusters K, with K varying from 1 to 10. The program was given no prior information on populations, and run under the admixture model with correlated allelic frequencies, with 100,000 MCMC iterations and a burn-in of 10,000 iterations.

Selfing rate was estimated using the maximum likelihood method of [START_REF] David | Reliable selfing rate estimates from imperfect population genetic data[END_REF], implemented in the software RMES. This method is not biased by the presence of null alleles. Tests for bottlenecks could not be performed because Hardy-Weinberg equilibrium was not met in most populations and for most loci [START_REF] Cornuet | Description and power analysis of two tests for detecting recent population bottlenecks from allele frequency data[END_REF]. Inter-population genetic distances DCE (Cavalli-Sforza & Edwards 1967) were computed using the software POPULATIONS [START_REF] Langella | Populations v[END_REF], and a neighbourjoining tree of populations was drawn after 100 bootstraps on individuals.

Results

Identification of hybrids and introgressed individuals

The alleles encountered in wild and domesticated populations were strongly differentiated [as already found by [START_REF] Duputié | Natural hybridization between a clonally propagated crop, cassava (Manihot esculenta Crantz) and a wild relative in French Guiana[END_REF]]. This excludes the possibility that historical gene flow between the two taxa could have blurred the relationships between populations of the wild taxon.

Individuals bearing alleles characteristic of domesticated cassava were encountered in three coastal populations: three individuals in MB bore one allele characteristic of domesticated populations, and 13 individuals in each of populations T1 and T2 exhibited one to three typical domesticated alleles. These 29 individuals had genotypes consistent with their being backcrosses of hybrids (F1 or later generations) with wild Manihot. They were removed from subsequent analyses (based on 344 individuals), together with locus GA57, which was fixed for a single allele in all remaining individuals.

Removing all populations where introgressed individuals have ever been found [T1, T2 and MB (this study), SM [START_REF] Duputié | Natural hybridization between a clonally propagated crop, cassava (Manihot esculenta Crantz) and a wild relative in French Guiana[END_REF] and MT (A. Duputié, unpublished results)] did not This is a postprint version of a paper published in Molecular Ecology (2009) 18:2897-2907; doi: 10.1111/j.1365-294X.2009.04231.x and available on the publisher's website.

change the conclusions (Supplementary Material).

Intra-population genetic diversity

A total of 36 alleles (4 -9 per locus) were encountered overall. Allelic richness AR, population fixation index f (estimator of FIS) and Nei's gene diversity H for all 14 populations are presented in Table 1. On average, allelic richness was higher in inselberg and western coastal populations (GM, MB, TP, SM) than in eastern coastal populations (Table 1; Mann-Whitney tests, inselberg vs. eastern populations, P = 0.010; western vs. eastern populations, P = 0.028, respectively). Over all populations, allelic richness ranged from 1.95 (for locus GA21) to 3.88 (for locus SSR68). Gene diversity followed the same pattern as allelic richness.

There was a strong overall heterozygote deficit, with f = 0.183 (95 % confidence interval: [0.106 -0.295]. Eight of the fourteen populations exhibited a significant deficit of heterozygotes. Both inland and coastal populations were affected. This deviation from Hardy-Weinberg equilibrium can be due to high selfing rates, to intra-population genetic structuring (Wahlund effect, notably in the inselberg populations, three of which are aggregates of populations found on different rocks), or to the presence of null alleles. High selfing rates are excluded by an using RMES software, as values (results not shown) in no case differed significantly from zero. Therefore, Manihot esculenta ssp. flabellifolia is mainly an outcrosser. Both latter explanations for heterozygote deficit are plausible. Indeed, population substructure at a very short spatial scale (less than 75 m) has already been observed in one population of this taxon (population SM, [START_REF] Duputié | Natural hybridization between a clonally propagated crop, cassava (Manihot esculenta Crantz) and a wild relative in French Guiana[END_REF]. Null alleles also are likely to be present, because the primers we used had initially been designed for domesticated cassava. Removing the locus exhibiting the highest frequency of null alleles did not modify the results presented here (Supplementary Material).

Population structuring

Population differentiation was high overall: θ = 0.363 (95 % confidence interval: [0.258 -0.427]). Pairwise FST values are given in Table 2, together with their significance. Most tests for population differentiation were significant. The only two large populations which were not differentiated were two inselberg populations (RT and WA).

Isolation by distance was significant when all populations were included (regression of FST / (1 -FST) with ln(distance), Mantel test after 10,000 permutations, 14 populations, P = 0.041; Supplementary Figure 1). However, as populations from Kourou (KP, CC) were very strongly differentiated from all other populations, irrespective of distance (note the high values of FST in Table 2), the isolation by distance pattern was much stronger when they were removed from the analysis (12 populations, P = 0.002; Supplementary Figure 1). Among inselberg populations, there was no evidence for isolation by distance (P = 0.795 but N = 4 populations; Supplementary Figure 1). Among coastal populations, isolation by distance was not significant when the populations from Kourou were included (regression of FST / (1 -FST) with geographic distance, 10 populations, Mantel test, P = 0.138), but was when these populations were removed (8 populations, P = 0.011; Supplementary Figure 1). [START_REF] Raymond | GENEPOP (Version 1.2) -Population genetics software for exact tests and ecumenicism[END_REF]; P-values < 0.05 are indicated in bold case). Population abbreviations are the same as in Figure 1. The optimal number of clusters formed by Bayesian clustering was four. Most individuals had a clear ancestry in one of the clusters (with STRUCTURE inferring that more than 90 % of their genome was drawn from this cluster). For each population, the average fraction of genome assigned to each of the four clusters is presented in Figure 2A. All inland individuals were assigned to cluster 1, except four individuals from Marouini. Coastal individuals from populations located west of Kourou fell into cluster 2, populations from Kourou were gathered in cluster 3, and populations from Tonate were classified in cluster 4. Individuals of populations SP and MT shared their ancestry between clusters 2, 3 and 4. When STRUCTURE was allowed to form a fifth cluster, these individuals remained of mixed ancestry. Individual assignments are presented in Figure 2B.

Table 1: Number of individuals N, allelic richness AR, gene diversity H and intra-population fixation index f for the 14 sampled populations and the associated P-value (exact test as implemented in GENEPOP

Inselbergs

From this analysis, we identified five genetic groups, four of which correspond to the clusters identified by STRUCTURE, the fifth one including the populations of unclear ancestry (SP and MT) (see map in Figure 3). Allelic richness, number of private alleles, f and Nei's genetic diversity for these five groups are presented in Table 3. Populations of the groups "Inselberg" and "West of Kourou" each bore five private alleles (i.e., alleles that were not encountered in any other group), whereas only one private allele was found in each of the groups "Kourou" and "Tonate", and none in the "Near Kourou" group. All groups except "Kourou" exhibited significant heterozygote deficits.

As tests for bottlenecks require Hardy-Weinberg equilibrium in the populations [START_REF] Cornuet | Description and power analysis of two tests for detecting recent population bottlenecks from allele frequency data[END_REF][START_REF] Luikart | Distortion of allele frequency distributions provides a test for recent population bottlenecks[END_REF], no tests were performed. Nevertheless, very low allelic richness in the populations from Kourou (KP, CC) and the fixation in these populations of a single allele at three loci that are polymorphic in other populations suggest that these two populations are issued from recent bottleneck events.

Coast-inselberg population differentiation

While STRUCTURE evidenced that coastal populations could be organised into four distinct genetic groups (one of which was of mixed ancestry), the population tree inferred from distances highlighted another aspect of genetic structure of the taxon in French Guiana (Figure 3). Inselberg and coastal populations were clearly separated: even though several groups were identified among coastal populations, all of them belong to the same genetic unit, and inselberg populations to another one. Among coastal populations, the two westernmost populations were basal, supported by a bootstrap value of 100 %.

Out of the 36 alleles we recorded, five were private to inselberg populations and 11 to coastal populations, further indicating a long isolation between inselberg and coastal populations. As most of these alleles were rare, smaller sample size in inselbergs than on the coast probably prevented the detection of some private alleles in inselberg populations. This is a postprint version of a paper published in Molecular Ecology (2009) 18:2897-2907; doi: 10.1111/j.1365-294X.2009.04231.x and available on the publisher's website. This is a postprint version of a paper published in Molecular Ecology (2009Ecology ( ) 18:2897Ecology ( -2907;;doi: 10.1111/j.1365-294X.2009.04231.x and available on the publisher's website.

Discussion

Hybridization and introgression between wild Manihot and domesticated cassava

Hybridization between the two taxa has already been shown to occur in several savannas from coastal French Guiana [START_REF] Duputié | Natural hybridization between a clonally propagated crop, cassava (Manihot esculenta Crantz) and a wild relative in French Guiana[END_REF]). Here, we found evidence for introgression of the wild taxon by domesticated genes in three further populations, all in the coastal savannas: T1, T2 and MB. These populations grow in human-altered environments (roadside, edge of a soccer field) and a few plants of domesticated cassava have been observed in their vicinity. Interestingly, no evidence for wild-crop hybridization was found in other zones of close sympatry between the wild and the domesticated taxa, such as in population KP.

Inselberg populations did not present any evidence of hybridization. On the one hand, this was not surprising, as inselbergs lie in very isolated areas, far away from any village. On the other hand, until recently the Amerindians, who have been the only inhabitants of inner French Guiana for the past centuries, moved their villages periodically. Therefore, some Amerindian villages could have been located at some time next to inselbergs where wild Manihot grew, or Amerindian farmers may even have planted wild plants together with cassava, as has been observed in recent times (J. Chave, pers. comm.). Gene flow would have been facilitated by such situations; however, if they occurred, neutral alleles issued from the domesticated taxon have disappeared since.

Evidence for past large savanna areas in southern French Guiana

All inselberg populations were classified in a single group by Bayesian clustering. Unexpectedly, differentiation was less marked within the group of inselberg populations than between inselberg and coastal populations, or among coastal populations. There was no isolation by distance between inselberg populations, which were very weakly differentiated.

This pattern could be explained by long-distance pollen or seed flow between inselberg populations, or from large source populations, which would homogenize French Guianan Manihot inselberg populations. However, longdistance pollen or seed flow seem inconsistent with the biology of the species and the patchy nature of the suitable habitats: inselbergs are often isolated from one another by several tens of kilometers. Further sampling of inselbergs, as well as analysis of Manihot samples from the neighbouring regions of Amapá, Suriname and Guyana, would help gain insight into the mechanisms by which it colonizes inselbergs.

Alternatively, shared ancestral polymorphism could explain the striking genetic homogeneity of inselberg populations: Manihot populations observed on inselbergs in the southern region could be remnants of formerly more extended, homogeneous populations. Connections between inselberg populations could have resulted either from the presence of large savanna areas, or from occasional largescale fires occurring in a mosaic of dry and gallery forest, creating labile and moving open areas where Manihot would have been able to grow. Several large-scale fire episodes have been recorded at various locations in French Guiana [START_REF] Tardy | Paléoincendies naturels, feux anthropiques et environnements forestiers de Guyane Française du Tardiglaciaire à l'Holocène récent -Approches chronologique et anthracologique[END_REF], even in zones continuously covered with rainforest [START_REF] Charles-Dominique | Forest perturbations and biodiversity during the last ten thousand years in French Guiana[END_REF]. Manihot populations from southern French Guiana, now found only on inselbergs, could therefore have been connected, at least periodically, either by a large savanna area, or by periodically disturbed dry forest areas. Demographic stochasticity, combined with genetic drift, may have led to local extinctions of small populations. Low frequency of gene flow -most probably through pollen -between inselbergs is nevertheless required to account for the homogeneity that is still observed between these inselberg populations.

Our hypothesis is consistent with the conclusions of Bush (1994), [START_REF] Dutech | 's website. and genetic diversity within French Guianan populations of a rainforest tree species, Vouacapoua americana[END_REF][START_REF] Noonan | Phylogeography and demography of Guianan harlequin toads (Atelopus): diversification within a refuge[END_REF][START_REF] Noonan | Phylogeography and demography of Guianan harlequin toads (Atelopus): diversification within a refuge[END_REF] that southern French Guiana must not have been a stable forest refugium area. Because only one species is studied here, the patterns that emerge from this study need to be confirmed by the study of other species, either restricted to open or dry environments, or to forest habitats. Further studies of population structure of other taxa restricted to inselbergs could help validate this scenario and yield further insights into past climate in the southern part of French Guiana.

Colonization of the coastal savannas

Population differentiation was much higher between inselberg populations and any coastal population than among inselberg populations. Similar coast-interior isolation has been documented for the forest tree Vouacapoua americana [START_REF] Dutech | 's website. and genetic diversity within French Guianan populations of a rainforest tree species, Vouacapoua americana[END_REF]) and for the poison frog genus Atelopus [START_REF] Noonan | Phylogeography and demography of Guianan harlequin toads (Atelopus): diversification within a refuge[END_REF]. As coastal savannas were formed only around 5,000 years BP [START_REF] Tissot | Evolution de la mangrove en Guyane au cours de l'Holocène: étude palynologique[END_REF], this raises the question of how they were colonized by Manihot. Although it does not include populations from outside French Guiana, the neighbor-joining population tree suggests that western coastal populations have been isolated for a longer time than eastern ones. Together with our findings of higher genetic diversity and higher number of private alleles in western than in eastern populations, this would be consistent with Manihot having colonized the coastal savannas from west to east, through a series of bottleneck events. Sampling of the neighbouring regions (Amapá, Suriname, Guyana and Venezuela) is needed to confirm this hypothesis. Significant isolation by distance among coastal populations indicates low gene flow between coastal Manihot populations (consistent with the hypothesis that long-distance gene flow is probably This is a postprint version of a paper published in Molecular Ecology (2009Ecology ( ) 18:2897Ecology ( -2907;;doi: 10.1111/j.1365-294X.2009.04231.x and available on the publisher's website. limited).

As inselberg and coastal Manihot populations are strongly differentiated, with no occurrence of the taxon on inselbergs north of Saül, and as the central part of French Guiana seems to have remained forested for the past 10,000 years [START_REF] Charles-Dominique | Forest perturbations and biodiversity during the last ten thousand years in French Guiana[END_REF], 2003), the central region probably remained somewhat wetter than the southern region, at least after the LGM [as has been proposed by de [START_REF] De Granville | Rain forest and xeric flora refuges in French Guiana[END_REF] and [START_REF] Dutech | 's website. and genetic diversity within French Guianan populations of a rainforest tree species, Vouacapoua americana[END_REF]]. Therefore, the colonization of coastal savannas by Manihot populations probably did not occur directly from the inselbergs inland, but rather from savannas located westwards. These "source" savannas could correspond to the Sipaliwini and Rupununi savannas of inland Suriname and Guyana (de Granville 1982).

Conclusion

Our data strongly support a long period of isolation between inselberg and coastal populations of Manihot esculenta ssp. flabellifolia. While inselberg populations are strikingly homogeneous for neutral genetic markers, coastal populations show a strong pattern of isolation by distance. As the hypothesis of ongoing gene flow from unsampled large source populations seems incompatible with the low gene dispersal abilities of the species, we hypothesize that inselberg populations of southern French Guiana were connected with one another until recent times, either through continuous savanna areas, or through periodically disturbed dry forest areas. The taxon appears to have colonized coastal savannas in the past few thousand years, from the western savannas of inner Suriname or Guyana, through a succession of bottlenecks. Although they should be completed by the study of other taxa, and of Manihot samples from savannas westwards, our results therefore argue in favour of the occurrence of formerly drier vegetation in the southern part of French Guiana, whereas the central and northern parts have probably remained forested since the LGM.

Figure 1 :

 1 Figure 1: Map of the sampling locations: filled squares indicate locations where Manihot was sampled; open squares indicate inselbergs where there was no Manihot. Unsampled inselbergs appear as dots. Numbers of individuals sampled are indicated in brackets. For the inland inselberg populations, when several close inselbergs were sampled, the numbers of individuals sampled on each is indicated after the total number for the population. GM: Grand Macoua; MB: Savane Mammaribo; TP: Trou Poissons; SM: Savane Manuel; CC: Carapa Hill; KP: Kourou Piste; SP: Savane des Pères; MT: Savane Matiti; T1: Tonate, population 1; T2: Tonate, population 2; RD: Roche Dachine; RT: Roche Touatou; WA: Wanapi; MA: Haut-Marouini. Grey areas correspond to altitudes > 200 m.

Figure 2 :

 2 Figure 2: (left) A) Map of the populations showing the average proportion of the genome inferred by STRUCTURE to be drawn from each of the four clusters. B) Proportion of the genome of each individual assigned to each of the four clusters. Each individual is represented by a vertical bar. Grey shades are as in panel A.

Figure 3 (

 3 Figure 3 (below): Neighbour-joining trees of populations (upper left) and groups of populations (lower left) based on distance (Cavalli-Sforza & Edwards, 1967). Trees are based on 100 bootstraps on individuals. Only bootstrap values > 50 % are shown. Abbreviations correspond to those in Figure 1. The five genetic groups identified by STRUCTURE are mapped (right-hand panel).

Figure 3 :

 3 Figure 3: Neighbour-joining trees of populations (upper left) and groups of populations (lower left) based on distance (Cavalli-Sforza & Edwards, 1967). Trees are based on 100 bootstraps on individuals. Only bootstrap values > 50 % are shown. Abbreviations correspond to those in Figure 1. The five genetic groups identified by STRUCTURE are mapped (righthand panel).

Table 2 :

 2 Pairwise FST values between all populations (lower-left matrix) and their significance (upper-right matrix, Benjamini & Hochberg's sharpened test, α = 0.05; NS: non-significant population differentiation).

	Coast (west to east)

Table 3 :

 3 Allelic richness AR, number of private alleles and total number of alleles PA/TA, gene diversity H, intra-population fixation index f and associated P-value (exact test performed in GENEPOP) for the five genetic groups of Manihot esculenta ssp. flabellifolia detected in French Guiana.

		Inselberg	West of Kourou	Kourou	Near Kourou	East of Kourou
		(MA, RD, RT, WA)	(GM, MB, TP, SM)	(CC, KP)	(SP, MT)	(T1, T2)
	AR	3.93	3.90	1.83	2.65	3.30
	PA / TA	5 / 25	5 / 27	1 / 11	0 / 15	1 / 19
	H	0.48	0.52	0.11	0.37	0.33
	f	0.27	0.18	0.03	0.42	0.08
	P-value	0.001	0.001	0.379	0.001	0.034

Acknowledgments

We are grateful to Jérôme Chave, Pierre Charles-Dominique, Olivier Hardy, Finn Kjellberg, Philippe Jarne, Hélène Joly and François Massol for critical reading of previous versions of this manuscript. Three anonymous reviewers helped us improve previous versions of this manuscript. We thank Guillaume Léotard and Benoît Pujol for the loan of Manihot samples, and Corinne Sarthou and Philippe Gaucher for collecting Manihot from inselberg populations during their expeditions to study other organisms. This study was funded by grants to D. McKey from various agencies of the French Government (Mission pour la Création du Parc de la Guyane, programme 'Écosystèmes Tropicaux' of the Ministry of Ecology and Sustainable Development, programme 'Impact des Biotechnologies dans les Agroécosystèmes' of the Ministry of Research and Contrat Plan État Région Guyane). A. Duputié was supported by a doctoral thesis grant from the French Ministry of Research. Genotyping was done in the 'Service Commun des Marqueurs Moléculaires en Écologie' of the CEFE, with the kind help of Laure Benoît and Hélène Vignes. Data used in this work were partly produced through molecular genetic analysis technical facilities of the IFR 119 "Montpellier Environnement Biodiversité".

Author Information Box

This work is part of Anne Duputié's PhD thesis, which aims at understanding some ecological and genetic aspects of the domestication of cassava. Marc Delêtre is a PhD student now working on cassava's genetic diversity and how people perceive and manage this crop in Gabon. Jean-Jacques de Granville is a botanist working as a senior researcher at the IRD centre in Cayenne, and Doyle McKey is a tropical biologist interested in coevolution, be it between humans and their crops, or between other kinds of organisms.


Sylvain Mondon
email: sylvain.mondon.auditeur@lecnam.net

Anne Marchais-Roubelat

Decision processes in action at sea, a methodological challenge for real world research

Keywords: Decision Making, Earth and Atmospheric Sciences, Uncertainty Management, Team and Organizational Factors in Complex Cognitive Work, Elite Sport

Relationships between decision process and action in real world conditions emerge as a key issue in elite sport as well as in organization studies. When pressure is high and environment is evolving rapidly, acting despite a limited situation awareness is common. In such stressed situations, the observation of phenomenon raises methodological difficulties such as the access to information and its validation with regard both to the decision-making process of the decision-maker and concurrently to the course of action. Studying in real time decision processes of a professional skipper at work during an ocean yacht race from the viewpoint of the router embedded in the skipper's team provides an opportunity to go forward in understanding relationships between decision processes and action in real world when phase shifts occur. The methodological choices are discussed and research directions for further real world researches are proposed.

INTRODUCTION

Relationship between decision processes and action in real world conditions emerge as a key issue in elite sport (Macquet & Kragba, 2015) as well as in organization studies (Marchais-Roubelat, 2012;McAndrew & Gore, 2015). During past decades laboratory approaches showed a variety of human behaviours and preferences in controlled experiments [START_REF] Kahneman | Maps of Bounded Rationality: Psychology for Behavioral Economics[END_REF]. Even if laboratory experiments can be very sophisticated as the one described by Brandt, Lachter, Battiste and Johnson for pilots (2015), some dimensions can't be replicated accurately enough to stand for real world. The full complexity of real world action involving multiple goals, time stress, uncertainty management, complex task is still not fully achievable in laboratory [START_REF] Kahneman | Conditions for intuitive expertise: A failure to disagree[END_REF]. In order to better understand relationships between decision processes and action, it is essential from our viewpoint, not to postulate anything about the relationship itself. Thus, we should be careful to incorporate no tacit premise in our approach. Especially, the framing of the research should not influence the way either decision processes or action are considered. A promising way to gain significant insights about relationships between decision processes and action is to work on real world by means of real time observation. Our guess is that stressed situations are likely to emphasise the relationships between decision processes and action because high time stress prevents from delaying acts. In such a stressed situation, it is common to act with limited situation awareness as shown by Klein, Orasanu, Calderwood and Zsambok (1993). The focus on real world practitioners adopted here meets the one promoted by naturalistic decision making (NDM) community for nearly 30 years [START_REF] Gore | Applications for naturalistic decision making[END_REF]. We have chosen a field relevant for both elite sport and organizational science: the action of skipper during ocean yacht race. It has been chosen because it consists of an organisation (skipper and his or her team) involved in an action where decisions must be made and implemented to handle the boat and manage its trajectory. So we can assume that during the race few decisions should be made in a given period of time. Our intention is to develop a fitted methodology to perform an inquiry in the field of yacht race in real time in order to better understand relationship between decision processes and action at sea with a view to improve training to get better performance. [START_REF] Gore | Applications for naturalistic decision making[END_REF] recalled that the area of elite sport is being investigated only for few years. So, our approach represents also an opportunity to explore a new field, highly compatible with NDM requirements, which could provide new "concrete examples from a specific profession" as recommended by Klein (2015). Even if "methodological advances in accessing expertise have gained respect and validity", NDM practitioners "recognize that the frameworks, models, and methods" used "have their limitations" [START_REF] Gore | Applications for naturalistic decision making[END_REF]. So, as we faced similar limitations, the developments proposed hereafter may be relevant for NDM practitioners. The methodological difficulties enhanced by NDM researchers are first highlighted when exploring the field in the context of action. Then, we discuss the design of a collaborative partnership for such a research during an ocean race, in terms of research embedment and of research methodological issues. First results and preliminary analysis are exposed. Finally, expected further outcomes are discussed and leads for further real world researches are proposed.

METHODOLOGICAL CHALLENGE OF ACCESS TO INFORMATION: ISSUES FOR OCEAN YACHT RACE DECISION AND ACTION PROCESSES

Developing a method aiming at performing a kind of "cognitive task analysis" [START_REF] Crandall | Working minds: A practitioner's guide to cognitive task analysis[END_REF] to elicit knowledge and analyse data in order to represent "real world" relationships between decision processes and action in the field of ocean yacht race raises several methodological difficulties. Those methodological difficulties form the challenge of accessing relevant information on a convenient way. This challenge is composed of several difficulties of different levels: duration, distance, acceptability, stakes and diversity of sources. Most of them have been dealt for other fields by NDM community in earliest studies for outdoors operations (Klein et al., 1993;Zsambok & Klein, 1997), for elite sport (Macquet, 2010, Macquet, Ferrand & Stanton, 2015) and for mountain expeditions [START_REF] Allard-Poesi | Performing Leadership "In-Between[END_REF].

Duration and phases of the action

Ocean yacht races usually last several days. Even if we expect some decision to be made in this period of time, the exact time of occurring can't be presumed. So the study must cover at least the entire period of the race not to miss any important phenomenon. Except in the case of mountaineering [START_REF] Allard-Poesi | Performing Leadership "In-Between[END_REF], most of the elite sports already studied by NDM practitioners were focused only on a short period of time. Furthermore, the sailors are 24h a day at work. Time is a key issue in organization (Orlikowski & Yates, 2002) as well as for elite athletes in managing competing activities (Macquet & Skalej, 2015). Sailors must dedicate their time to competing tasks like tuning, steering, computing route, analysing situation, observing environment, communication to media, checking the boat, repairing broken pieces or sleeping. The researchers must be ready to capture phenomenon which can occur at any time during any activity (Klein et al., 1993). Because of its duration, ocean race as an action process includes many decisions, which can be connected with different phases of the race. As a result, the connection between these decisions and phase shifts in the action appear key issues for research in the field.

Distance to the action

In the case of sailing across an ocean, the size of natural field is only limited by continents and islands. Keeping a continuous access to action wherever the boat is, is a difficulty as soon as the boat leaves the dock. The idea to shorten distance between action and researcher is a leading idea in the earliest studies developed by NDM community about fireground commanders (Klein et al., 1993). The issue is to be as close as possible to the action where decision are supposed to be made in order to get relevant information for subsequent analysis and interviews by performing direct observation in real time if possible. When the distance becomes a problem, means of communication may compensate it as exposed by Orasanu and Fisher (1997) in the field of spaceship crew and by [START_REF] Allard-Poesi | Performing Leadership "In-Between[END_REF] in the field of mountaineering. Those studies both show that means of communication (emails and phone calls) are essential to keep researchers in touch with practitioners when they can't be at the same place. During an ocean race, although information technology shortens the distance between the sailor and his or her team, the skipper has a full autonomy of decision to influence the course of the action.

Awareness of what is at stake

The professional skippers of the biggest modern yachts crossing ocean and rounding the world are very experienced sailors. They are considered as experts by others in the sense given by Klein (Klein, 1998). In such a technical complex field, being aware of what is at stake for the expert is a difficulty for the researcher. This is all the more critical when action is going on because direct interview can't be dedicated to long explanation. The researcher faces also the difficulty of knowing what are the meaningful cues to be noticed and how to seize them. The issue of understanding what is at stake is an essential point to get and validate information and to avoid later misinterpretation (Klein et al., 1993). For ocean race, four main domains of interest can be identified to define what is at stake: boat handling, weather conditions, information and telecommunication systems, competition evolution.

Distributed situation awareness

During yacht race, information can be collected from a lot of different sources. Each source provides data from specific viewpoint useful to document at least one side of the action. Describing the side of the environment can be done by quantitative data as well as by qualitative data. Quantitative raw data can be provided by measurements (onboard sensors, remote sensing, buoy, weather stations) or by model analysis (national weather services). Qualitative data can be provided by weather bulletin, comprehension of the router, perception of skippers. Describing what is happening in term of action at race level can be informed by general trajectories of the whole fleet provided by race organizer, by detailed trajectory of a given boat provided by one team and by high frequency onboard monitoring of the route. These quantitative data sets can be completed by qualitative statements and comments from individuals or organizations (ex. press release). Taking into consideration "both human and technical agents as well as the way they interact" is the basis of distributed situation awareness (DSA) described by Stanton (Stanton, 2016). DSA is a critical issue for the athletes and coaches (Macquet & Stanton, 2014) and also for researcher analysis (Macquet, 2013;Macquet et al., 2015). In ocean race, data provide a double perspective. On the one hand, data serve as a decision support system to help the sailor to make decisions to create new phases in the action processes. On the other hand, data are benchmarks for researchers to assess the decision process and action.

Acceptability of the research

Another crucial point for researcher interested in "real world" is to make the research acceptable for the field he or she would like to study. To that respect, the first point to deal with is to get an agreement in principle from a skipper to be studied at work in the course of action during a yacht race. As previously recalled time management is a critical point for skipper, the researcher position can't be too much intrusive. The difficulty for the researcher is to propose a research design light enough to be agreeable to the skipper and deep enough to enable fruitful inquiry: a balanced position must be negotiated. The purpose of the researcher is to have an access to relevant information in a transparent manner without significantly disturbing the way action is taking place.

DESIGNING A COLLABORATIVE PARTNERSHIP: PROCESS AND DISCUSSION

The research project intends to better understand the relationship between decision processes and action during ocean yacht race run by experienced sailors skipping multihull boats. Thanks to an 11 years experience in advising skippers between 2000 and 2011 in weather forecasting and routing, one of the two researchers involved in the project has a privileged access to the field. An agreement in principle has been given by one racing team to open the doors to a research project over several years. Since this point of departure in 2012, we have been progressively working out a convenient methodology to keep cautiously mutual trust and information exchange. We are presenting important issues encountered between 2012 and 2016 when the approach has been developed along with preparation, training and races ("Route du Rhum 2014" and "Transat Jacques Vabre 2015"). Then the focus is put on the next race "Transat Jacques Vabre 2017" for which we intend to implement an intensive observing period to highlight specific settings.

Embedded research

Even necessary, agreement in principle is not sufficient to perform an in depth inquiry. To our comprehension from preliminary discussions, the key issue is that the research has to be not only acceptable, but also promising enough for the skipper to sound useful in one way or another for short and long term. The time spent for the research from the viewpoint of the skipper is seen as a long term investment with regard to the expected outcome of the research. The second step is then to assess different possible positions for the researchers to do proper in depth research in a transparent manner and a convenient way for the team. Considering short term, the researchers and skipper acknowledged that the main challenge is to compensate in one way or another the fact that the sailors or team could spend time by discussing with researchers instead of paying attention only to they work in the course of action. Thus the deal concluded to that respect is that the researcher who is skilled in weather forecasting and routing can have a full access of the work of the team in real time in the condition that his or her skills can benefit to the team if he or she detects an occasion to do so. It is a kind of short term investment. The skipper expects that the skills of this researcher can save time to handle situation and complement distributed situation awareness. By doing this, the researcher is accepted as a special team member. Let's notice these short term as well as long term investments have no guaranty of success, they both are risky gambles for the skipper and for the researcher. Thanks to such a unique opportunity to be part of team, the research project is designed with one embedded researcher only whereas the second one keeps a distant outside viewpoint of the field and focuses on methodology and analysis, from common theoretical bases. The insider position is then balanced with the outsider one to reduce as much as possible biases in data collection as well as in subsequent analysis.

Time and ethics

Being inside a team is likely to have an influence on the action management. So there is a significant risk of disturbance and change in the focus of the sailors that has to be taken into account by the researcher. To that respect, our intention is to cover different stages of the preparation of the race by a compatible research activity so as to benefit each opportunity to learn more about the field and the skipper as well as to build trust all along the research project. It seems to us that the skipper should start the race (and we should start our intensive observation as well) with a mind free to focus entirely and deeply to his work (and we should do it as well). Furthermore, we would not accept a situation where our research is responsible for even the smallest trouble in the mind of the skipper. All discussions prior to the race must avoid to induce discomfort neither for the skipper nor for his or her team. This point is essential to our approach in order to neither influence the way of thinking nor the way of doing things of practitioners. This revealed to be crucial to create condition of acceptability of the research. To avoid any trouble four commitments have been made. The first commitment is to assure that the researchers behaviour will be as low profile as possible. The second commitment is to share the results with the sailors, as soon as they are robust enough. The third commitment is not to communicate personal information publicly and not to convey any information to other teams. The forth commitment is to mention any information in case it could be relevant for the race to the skipper, the router or the technical team.

Capacity and awareness

We have been implementing different tools and methods since 2013, bearing in mind the advice of Crandall et al. (2006, p. 143) by "understanding the way people think and reason in natural contexts, cognitive task analysis practitioners are more likely to recognise important aspects of cognition when they encounter them". Our method provided us a training of about four years in the field by observing action, interviewing practitioners, data collecting and analysis of events. We progressively developed a deeper understanding of stakes, multiple goals by discussion and interview, direct observation on board and in the shipyard. By doing this we validated the usefulness of the method and tools for the field and built continuously a trustworthy climate for collaboration. We now significantly better understand what's happening, what is at stake and the research is accepted by the skipper, the team and the sponsor. The researcher in reflexive position inside a team shares shemata and common knowledge of the team (Lipshitz & Ben Shaul, 1997) and is sensitized to collective mind (Weick & Roberts, 1993).

Intensive observing period

We intend to implement an intensive observing period for the next east to west double handed transatlantic involving "Classe Ultime" yachts (Trimarans longer than 60 feet) due to start from Le Havre (Normandie, France) in October 2017 to Salvador de Bahia (Brazil). As routing is allowed in race rules, co-skippers are going to work with a router to define and adjust race strategy. To be as close as possible to the practitioners without interfering in the race process, one researcher is shadowing the router. In this position the researcher will have real time access to all technical information. Furthermore, he or she will be able to hear and record discussions between skipper and router during the race and days before start. He or she also will be able to interview router as often as needed in the course of action and at times he may have an access to direct discussion with skipper to clarify point as well as with sponsor, technical team and communication agency. As the route of the race crosses Atlantic Ocean starting at mid latitudes and finishing in the tropics of southern hemisphere (Brazil) the sailors are facing typical navigation problems. Indeed, they must sail at first in the westerly flow of mid latitudes then find a way to catch the northeasterly trade winds of northern hemisphere. Then they must cross the doldrums and catch the trade the southeasterly trade winds of southeast hemisphere, which creates phase changes. As several weather systems are encountered, a proper management of the transition from the influence area of one to the influence area of the next one is a crucial issue. So not only decision are to be made, but important decisions are to be made at least at those major transitions.

Dynamic and complex field

Modern multihull yachts of "Classe Ultime" are the fastest sailing boats (speed are faster than 40 knots) having a race across ocean. In that case, those boats can move faster than most of usual weather systems on the one hand and can sail twice faster as the wind speed in particular conditions. Taking this into account, the sailing area is very large because an extra distance can be more than compensated by a small increase in wind speed or a better angle in wind direction. As biggest and fastest boat, "Classe Ultime" yachts are the one which are concentrating interest of media and investment of sponsors. The teams are composed of highly skilled workers, the crew are composed of experienced professional skippers and sailors. Skippers have to manage budget, boat, team, crew, innovation and are used to make decisions in stressed and uncertain situation at sea. Situation is dynamic because of three reasons: natural conditions (both atmosphere and ocean) are evolving in a continuous way, the boat is moving along its trajectory on its own pace, and rivals are sailing their way too. These yachts require high skill to be skipped properly. All the skippers of "Classe Ultime" trimaran have been sailing multihul for at least 10 years and up to 20 years for most of them. As an example, in particular conditions, the difference between high performance and danger for life (capsize) is less than 20 centimetres along a rope or less than 5 degrees of the true wind angle. The racing team is managed by one of the most experienced skipper (Lionel Lemonchois). He has been sailing for more than 35 years. During the last 15 years the sailor broke more than 10 world records in trans-oceanic route or round the world non-stop on big catamarans and trimarans. He is considered as one of the most experienced multihull skippers by others.

RESULTS AND PRELIMINARY ANALYSIS

Along the development of methodological framework, different embedded observations have been performed during last 4 years. Action based observation and preliminary analysis showed that phase shifts are important issues in the relationship between decision processes and action. Preliminary analysis is based on 3 cases involving the same skipper: crew training (2 days onboard and workplace observations), single handed transatlantic race (10 days observation inside the shoreteam in a routerlike position), double handed transatlantic race (10 days observation inside the shoreteam shadowing the router). Decision processes in those cases are performed in stressed situations especially high for the two race cases. From the analysis at the level of the action focused on the boat considered as an organization, we can assume that the way action is going on is governed by rules. Those rules depend either on the environment only (ex. weather, overarching action, side action), or on the organization only (ex. boat capacity, crew skills, team behaviour, expected goal), or on an interaction between environment and the organization (ex. the way low pressure is pulling a sail to create aerodynamic power, interaction between skipper and race organizer). A shift in phase occur when at least one rule is modified for a significant period of time according to length of action. If it seems quite easy to identify and describe one phase or another, the shift between them is much more complicated than it could be inferred at first guess. Embedded observations showed that a phase shift can spend quite a long time. Influence of a given rule can decrease gradually whereas another one is increasing. The influence of a given rule can fluctuate in a general trend of decrease or increase. It has been noticed that all along a transition period between two well settled phases, there is a gap in analysis of relation between action and decision if the influence of changing rules (emerging or disappearing) is not taken into account. Our guess is that such an approach focused on transition periods may benefit, not only to NDM practitioners, but also to organizations for performance improvement.

CONCLUSION AND RESEARCH DIRECTIONS

As skippers are convinced that human factor can make a difference in yacht race there is an opportunity to design a research project to study this field to better understand relationship between decision processes and action in "real world" to improve performance. Thanks to a specific skill held by one researcher in an important domain for the performance of the action, it has been possible to enter the field with mutual trust and respect. Considering longer term than a single race, skippers rely on researchers also to help them to improve their performance for next stages of their career. In terms of acceptability of the research as well as in terms of capacity building of the researcher, it appears to be all the more important for the researcher to be involved in the team as early as possible. Discussion showed that to be accepted by the team the researcher activity must not interfere the course of action and not modify the way of thinking of skippers during the race. The researchers are expected not to disturb the organisation even if the only fact that a research is performed can have an impact on the team behaviour at work. The design of intensive observing period lasted four years because of cautious step by step working out to find the deepest acceptable way to do research in embedded but not intrusive position. The first outcome we were expecting was to show that the original position of the researcher can provide valuable information unreachable otherwise. Second, we expect to identify pieces of information that can lead to better understand relationships between decision processes and the changes occurring in the action process. We are also expecting to show how rich a qualitative empirical analysis of real world action can be when conducted in such dynamic context documented by very different sources of information. Finally, we are expecting to identify tracks to follow to better understand how expert sailors are thinking at sea and how they can improve their ability to make a difference in real time. As our approach is relevant to macrocognition (Klein, Hoffman & Militello, 2016) expected outcomes may lead to refine NDM frameworks and applications in a multidisciplinary effort according to the views expressed by Mosier and Militello (2016). Complementary research involving embedded position in other fields could be developed. The proposal is to do more researches as an insider to identify small scale processes in real time as close as possible of the action in the real world. The way we conduct our research project could be implemented in other organizational contexts in order to look for similarities or differences in the relationship between decision processes and action. A comparison of knowledge elicitation in real world studies by the method proposed here and by other methods like ethnomethodology, action research or process studies could identify methodological advantages of each for a better understanding of the relationship between decision processes and action in different uncertain contexts where phase shifts occur. Finally, for each step forward it would be interesting to confront the fields results to existing decision making models.

ACKNOWLEDGMENTS

We gracefully thank sailors, teams and sponsors for their collaboration and the time they dedicated to our work.

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