Lucas Mazal
email: lucas.mazal@uca.fr

Dov Corenblit

Nadia Barsoum

Johannes Steiger

Leif Skøt

Boris Fumanal

Irène Till-Bottraud

Leif Skot

Irène Till- Bottraud

Guillaume Leroux

Véronique George

Vanina Guerin

Charles Poncet

Lydia Jaffarelo

Fine-scale spatial genetic structure and intra-specific interactions of Populus nigra within a natural river corridor along the lower Allier River (France)

Keywords: Black poplar, Clonality, Density-dependent processes, Facilitation, Microsatellites, Population genetics

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

Introduction

Spatial genetic structure (SGS), defined as the non-random spatial distribution of genotypes, characterizes the relationship of relatedness estimates between pairs of individuals and their physical proximity [START_REF] Loiselle | Spatial genetic structure of a tropical understory shrub, Psychotria officinalis (Rubiaceae)[END_REF][START_REF] Hardy | Estimation of pairwise relatedness between individuals and characterization of isolation-bydistance processes using dominant genetic markers[END_REF] with positive spatial genetic structure commonly interpreted as a sign of restricted gene flow. Thus, patterns of dispersal of pollen and seeds have important consequences for the SGS [START_REF] Banks | How does ecological disturbance influence genetic diversity?[END_REF].

However, density-dependent, biotic interaction processes may alter the SGS of plant populations generated by dispersal. For example, the thinning process in maturing forests may change their genetic composition and structure by removing groups of siblings [START_REF] Yoda | Self-thinning in overcrowded pure stands under cultivated and natural conditions[END_REF][START_REF] Harper | Population Biology of Plants[END_REF]. Biotic interactions are likely to change over life stages, especially when the environment changes as well. The stress gradient hypothesis [START_REF] Bertness | Positive interactions in communities[END_REF] predicts that different stress levels will change the type of interaction between individuals from positive (i.e. facilitation in stressful environments) to negative (i.e. competition in benign environments). As most plants are not capable of movement, they will be in contact with their direct neighbours throughout their lifetime, and plant interactions occur where plants overlap their 'zones of influence', e.g. among immediate neighbours at fine spatial scales [START_REF] Stoll | A Neighborhood View of Interactions among Individual Plants[END_REF]. In addition, the SGS pattern could in turn influence what type of biotic interaction will take place among the immediate neighbours on plants [START_REF] Fajardo | Disturbance and density-dependent processes (competition and facilitation) influence the fine-scale genetic structure of a tree species' population[END_REF]. Few studies have linked intra-specific interactions and the pattern of SGS within populations [START_REF] Till-Bottraud | Multi-stemmed trees of Nothofagus pumilio second-growth forest in Patagonia are formed by highly related individuals[END_REF][START_REF] Segovia | On the advantage of sharing a holdfast: effects of density and occurrence of kin aggregation in the kelp Lessonia berteroana[END_REF]. Studies of SGS in plants generally address large geographic areas [START_REF] Hardy | Fine-scale genetic structure and gene dispersal inferences in 10 Neotropical tree species: Gene dispersal in tropical trees[END_REF][START_REF] Kettenring | Fine-scale genetic diversity and landscapescale genetic structuring in three foundational bulrush species: implications for wetland revegetation: Bulrush genetic diversity and structuring[END_REF][START_REF] González-Robles | Spatial genetic structure of a keystone long-lived semiarid shrub: historical effects prevail but do not cancel the impact of recent severe habitat loss on genetic diversity[END_REF] and sampling often neglects the scale of the "zone of influence" of the studied species. Over smaller areas, SGS is often referred to as "fine scale genetic structure" (FSGS), but the smallest distances studied vary greatly among plant types. In herbaceous plants, studies are at the scale of metres [START_REF] Ning | Contrasting finescale genetic structure of two sympatric clonal plants in an alpine swampy meadow featured by tussocks[END_REF][START_REF] Loh | Fine-scale spatial genetic structure, neighbourhood size and gene dispersal in clonal plants: exploring the best possible estimates[END_REF] whereas in trees it is usually the scales of kilometres [START_REF] Sagnard | Effects of seed dispersal, adult tree and seedling density on the spatial genetic structure of regeneration at fine temporal and spatial scales[END_REF][START_REF] Born | Smallscale spatial genetic structure in the Central African rainforest tree species Aucoumea klaineana : a stepwise approach to infer the impact of limited gene dispersal, population history and habitat fragmentation: SGS in a central african tree species[END_REF][START_REF] Deng | Conservation implications of fine scale population genetic structure of Ficus species in South African forests[END_REF]. In the past few years, FSGS studies in trees have been conducted on smaller scales [START_REF] Kitamura | Distinctions in Fine-Scale Spatial Genetic Structure Between Growth Stages of Picea jezoensis Carr[END_REF][START_REF] Twyford | Multi-level patterns of genetic structure and isolation by distance in the widespread plant Mimulus guttatus[END_REF] but little is known at the scale of a few meters or less. In the following, we will use "fine-scale spatial genetic structure" (FSGS) to refer to scales including the distance at which individuals interact with each other (i.e. including the zone of influence).

Populus nigra L. (Salicaceae), the black poplar, is a pioneer tree growing in natural river corridors in Europe. It is considered as a priority for research and conservation because human activities have severely reduced population sizes and increased population isolation [START_REF] Lefèvre | Strategies for the conservation of a pioneer tree species Populus nigra L. in Europe[END_REF][START_REF] Villar | Ressources génétique du peuplier noir (Populus nigra L.) dans la plaine rhénane alsacienne : états des lieux, programme de conservation et perspectives[END_REF]. Additionally, poplar populations in their early life stages (<15 years) can have a significant effect on hydrogeomorphological processes, fluvial landforms and related habitat conditions by trapping huge amounts of fine sediment, nutrients and organic matter [START_REF] Hughes | Allocation of River Flows for Restoration of Floodplain Forest Ecosystems: A Review of Approaches and Their Applicability in Europe[END_REF]Corenblit et al., 2016a). This ecosystem engineering effect (sensu [START_REF] Jones | Organisms as Ecosystem Engineers[END_REF] is maximised where saplings grow in high densities. Sediment and organic material accretion improve resistance to mechanical stress but also improve poplar survival and growth [START_REF] Corenblit | The biogeomorphological life cycle of poplars during the fluvial biogeomorphological succession: a special focus on Populus nigra L.: The biogeomorphological life cycle of poplars[END_REF]. Intraspecific facilitation is supposed to be a major driver at the establishment stage in densely populated recruitment bands or patches [START_REF] Barsoum | Relative contributions of sexual and asexual regeneration strategies in Populus nigra and Salix alba during the first years of establishment on a braided gravel bed river[END_REF][START_REF] Corenblit | The biogeomorphological life cycle of poplars during the fluvial biogeomorphological succession: a special focus on Populus nigra L.: The biogeomorphological life cycle of poplars[END_REF][START_REF] Corenblit | Niche construction within riparian corridors. Part II: The unexplored role of positive intraspecific interactions in Salicaceae species[END_REF], and may correspond to a positive niche construction effect sensu Odling-Smee et al. (2003). In this context, the fine scale genetic structure could provide valuable information on the colonization process of alluvial bars by P. nigra within the fluvial corridor. Moreover, the feedbacks between FSGS and intra-specific interactions and their impact on seedling establishment and population genetic diversity and structure remain unexplored.

The aims of this study were to understand local patterns of dispersal and intra-specific interactions under natural conditions during the early life-stages of P. nigra. To do so, we explored the FSGS (at a meter scale or less) of three different cohorts of black poplar over a 1.6 km reach of the Allier River (France) located in a nature reserve. We used microsatellite markers that allowed us to estimate the genetic diversity of the population. As vegetative propagation was identified in the population, we investigated its geographic distribution, in particular in relation to topography using a digital elevation model (DEM) of this reach of the Allier River. The originality of the present study lies in the very fine scale (metric) genetic structure approach combined with the comparison between different cohorts.

Material and Methods

Study Species

Populus nigra L. (Salicaceae) is a fast-growing tree species with a good tolerance to submersion, sediment burial and high temperatures [START_REF] Chamaillard | Efficience de l'utilisation de l'eau chez le peuplier noir (Populus nigra L.) : variabilité et plasticité en réponse aux variations de l'environnement. Biologie végétale[END_REF]. The species is dioecious and wind pollinated. Seed dispersal combines a wind-mediated phase often followed by a secondary hydrochorous phase [START_REF] Barrat-Segretain | Strategies of reproduction, dispersion, and competition in river plants: A review[END_REF]Karrenberg and Suter, 2002). The seeds are short-lived (they cannot survive more than a few weeks), and germination only occurs immediately after arrival on suitable bare moist alluvial bars [START_REF] Barsoum | Regeneration response of black poplar to changing river levels[END_REF]. As a pioneer species, the eco-hydrological conditions for regeneration include freshly disturbed, competitor-free sediments.

Prime seedling recruitment habitat in the active alluvial zone occurs at the immediate margins of the main or secondary channels and is highly dependent on water table level fluctuations [START_REF] Corenblit | The biogeomorphological life cycle of poplars during the fluvial biogeomorphological succession: a special focus on Populus nigra L.: The biogeomorphological life cycle of poplars[END_REF]. Harsh summer (drought) and winter (submersions, erosion and/or burial) conditions on alluvial bars allow seedlings to establish only during restricted time periods, not necessarily every year, and on areas of the alluvial bars that are close enough to the water resource to permit the roots to track the water table during the dry season (i.e. recruitment box sensu [START_REF] Mahoney | Streamflow requirements for cottonwood seedling recruitment-An integrative model[END_REF][START_REF] Barsoum | Regeneration response of black poplar to changing river levels[END_REF][START_REF] Guilloy | Abrupt drops in water table level influence the development of Populus nigra and Salix alba seedlings of different ages[END_REF][START_REF] Stella | How do riparian woody seedlings survive seasonal drought?[END_REF].

Suitably positioned seedlings on alluvial bars near the main channel generally form more or less elongated recruitment bands or patches composed of individuals of the same age (a cohort). Such spatial patterns suggest that a significant proportion of seeds are transported by the river. Following the dispersal and early establishment phase, subsequent sapling development (first 3 years according to [START_REF] Cooper | Factors controlling the establishment of Fremont cottonwood seedlings on the Upper Green River, USA[END_REF] is governed by strong feedback mechanisms between plants and hydro-geomorphological processes (i.e. water flow, sediment transport and landform construction). P. nigra is additionally capable of vegetative reproduction: one individual can produce multiple shoots, or ramets, which all share the same multilocus genotype (MLG, sensu [START_REF] Arnaud-Haond | Standardizing methods to address clonality in population studies[END_REF]. From here on, we will use ramets to refer to the different shoots originating from one individual MLG. In highly dynamic rivers, vegetative reproduction can be a significant alternative mode of recruitment [START_REF] Barsoum | Variations in levels of clonality among Populus nigra L. stands of different ages[END_REF][START_REF] Francis | The survival and growth response of Populus nigra fragments to differing hydrogeomorphological conditions[END_REF] and can complement sexual reproduction in colonizing new areas [START_REF] Tinschert | Alternate reproductive strategies of Populus nigra influence diversity, structure and successional processes within riparian woodlands along the Allier River, France[END_REF]. However, vegetative reproduction is reported to be a primarily local strategy, especially in disturbed environments [START_REF] Arens | Molecular genetic analysis of black poplar (Populus nigra L.) along Dutch rivers[END_REF]Smulders et al., 2008).

Study site

The study site was located along the lower reaches of the Allier River in the Réserve Naturelle Nationale du Val d'Allier, near Châtel-de-Neuvre (Fig. 1; 46°25'06.5"N, 003°19'43.2"E; 220 m a.s.l.) in central France. Contrarily to most European rivers, the flow regime along the lower reaches of the Allier River is largely unregulated and morphodynamics within the Nature Reserve are only moderately impacted by human activities [START_REF] Garófano-Gómez | Vegetation succession processes and fluvial dynamics of a mobile temperate riparian ecosystem: the lower Allier River (France). Géomorphologie: relief, processus[END_REF]. Bank erosion in the outer bends of meanders and recurrent active channel migration promote the formation of bare gravel bars suitable for P. nigra regeneration. The progressive shifting of the river channel allows for periodic recruitment episodes, resulting in different aged cohorts of P. nigra recruits growing in elongated bands parallel to the main channel [START_REF] Garófano-Gómez | Vegetation succession processes and fluvial dynamics of a mobile temperate riparian ecosystem: the lower Allier River (France). Géomorphologie: relief, processus[END_REF].

Sampling strategy

We sampled same-age individuals over very short distances within recruitment bands. Three gravel bars were selected for this study along a 1.6 km reach of the Allier River. We sampled 21 groups of 15-20 individuals. This sampling strategy ensured that we incorporated a broad range of distances between individuals: from a few centimetres to 1.6 km (Fig. 1). The mean distance between individuals from a same patch was 12.33 m. Each individual was georeferenced in the field using a GPS Trimble GEO7X™ with a decimetric precision. Groups of individuals were categorised into three age cohorts: 'young' (less than 5 years old), 'middle-aged' (approximately 10 years old) and 'old' (more than 20 years old), based on their appearance in aerial photographs (we used all the available aerial photographs, i.e. 1992, 1997, 1998, 2002, 2009, 2013 and 2016). During the sampling period in November and December 2017, we collected cuttings for DNA extraction and tree genotyping since no leaves were available in winter. We collected cuttings from a total of 28 'young' individuals, 276 'middle-aged' individuals and 44 'old' individuals (total of 348 individuals sampled). Cuttings were wrapped in wet tissue and stored in the laboratory at 4°C for 3 weeks prior to DNA extraction.

DNA extraction and genotyping

Cambium fragments were collected from the cuttings in the laboratory and stored in silica gel to promote rapid tissue desiccation, thereby avoiding DNA degradation. We used 8 unlinked codominant microsatellites (SSR) markers to genotype individuals: WPMS13, WPMS22 [START_REF] Smulders | Trinucleotide repeat microsatellite markers for black poplar (Populus nigra L.): Primer Note[END_REF], PMGC2385, PMGC93, PMGC2578, PMGC14, GCPM2995 and ORPM221 [START_REF] Chenault | SSR-based analysis of clonality, spatial genetic structure and introgression from the Lombardy poplar into a natural population of Populus nigra L. along the Loire River[END_REF][START_REF] Faivre-Rampant | New resources for genetic studies in Populus nigra : genome-wide SNP discovery and development of a 12k Infinium array[END_REF]. Markers were arranged in 2 multiplexes. Multiplex1: ORPM221, GCPM2995, WPMS13, WPMS22; Multiplex2: PMGC93, PMGC2385, PMGC2578, PMGC14. We used indirect tagging with M13-tailed primer method [START_REF] Oetting | Linkage analysis with multiplexed short tandem repeat polymophisms using infrared fluorescence and M13 tailed primers[END_REF]. In this method, instead of synthesizing one specific fluorescently labelled primer for each SSR marker, only a dye-labelled M13 primer is needed.

The polymerase chain reaction was carried out in a volume of 10 µL, which contained 2.5 µL of DNA (at 10 ng/µL) and 7.5 µL of the following mix: 5 µL Master mix (AmpliTaq Gold™ 360), 0.5 µL primer mix (labelled with M13 fluorochromes Applied Biosystems™ 6-FAM, NED, PET and VIC), 0.05 µL tagged M13 primer and 1.95 µL H2O. The profile of the touchdown phase with 7 cycles was as follow: 95 °C for 30s, 62 °C for 30s and 72 °C for 30s, with a temperature reduction of -1 °C per cycle. The 30 classical PCR cycles were as follow: denaturation at 95 °C for 30s, annealing at 55 °C for 30s and extension at 72 °C for 30s. The M13 tail and fluorochromes were bound during 8 cycles with the following thermal profile: 95 °C for 30s, 56 °C for 30s and 72 °C for 30s. The final extension step was 72 °C for 5 min. The PCR products were run in an automatic sequencer (96 capillars 3750xl DNA Analyser Applied Biosystems™). Electropherograms were analysed using GeneMapper™ v.5. To test reproducibility, 16 randomly chosen individuals were replicated in the genotyping. No error was detected.

Genetic analyses

Two individuals were removed because of missing data for two loci. Loci were tested for null allele frequencies with Brookfield's methods [START_REF] Brookfield | A simple new method for estimating null allele frequency from heterozygote deficiency[END_REF]) using Microchecker v2.2.3 (Van Oosterhout et al., 2004). The GCPM2995 marker presented a high null-allele frequency (Table 1) and was therefore removed from the dataset.

To estimate the discrimination power of the dataset, we calculated the probability of sampling two different genotypes with the same multilocus SSR phenotype (probability of identity) with one to seven markers using GenAIEx v6.5 [START_REF] Peakall | GenAlEx 6.5: genetic analysis in Excel. Population genetic software for teaching and research--an update[END_REF].This probability decreased from 9.9 e10-2 to 4.6 e10-3 with one marker (depending on the marker), and reached a plateau around 3 e-07 with five markers suggesting a high discrimination power of our seven markers combination (see supplementary material Fig S1). Clones were identified using the "Multilocus matches" procedure from GenALEx.

Genetic diversity

As high levels of clonality can have a significant impact on SGS because some multilocus genotypes can be overrepresented over small distances [START_REF] Chenault | SSR-based analysis of clonality, spatial genetic structure and introgression from the Lombardy poplar into a natural population of Populus nigra L. along the Loire River[END_REF], we kept only one ramet per multilocus genotype when two or more ramets were present, resulting in a dataset of 280 multilocus genotypes. We estimated allele frequencies, total and effective number of alleles, expected and observed heterozygosities and inbreeding coefficient using SPAGeDi 1.5 software [START_REF] Hardy | spagedi: a versatile computer program to analyse spatial genetic structure at the individual or population levels[END_REF]. Genetic structure was analysed using the STRUCTURE 2.3.4 software [START_REF] Pritchard | Inference of population structure using multilocus genotype data[END_REF], with 20 runs, a burnin period of 100 000 iterations followed by 200 000 additional MCMC iterations, with an admixture model, uncorrelated allele frequencies and recessive alleles. The best number of clusters (K) was estimated using the ΔK method of [START_REF] Evanno | Detecting the number of clusters of individuals using the software structure: a simulation study[END_REF] and visually verifying the highest values of LnP.

Spatial genetic structure

SGS was assessed for the whole population and within sampling groups (FSGS) for each age cohort using SPAGeDi 1.5 software [START_REF] Hardy | spagedi: a versatile computer program to analyse spatial genetic structure at the individual or population levels[END_REF]. Loiselle multilocus kinship coefficients, Rij [START_REF] Loiselle | Spatial genetic structure of a tropical understory shrub, Psychotria officinalis (Rubiaceae)[END_REF], were calculated between each pair of individuals. Because Rij is a kinship coefficient relative to the population mean, negative values can result, meaning that two individuals are less related on average than randomly selected individuals from the population [START_REF] Hardy | Estimation of pairwise relatedness between individuals and characterization of isolation-bydistance processes using dominant genetic markers[END_REF]. Kinship coefficient values were averaged within distance classes (d), giving F(d) and plotted against geographical distances. Ten classes were formed manually.

Significance was tested by running permutations of the spatial position of individuals 10,000 times, yielding a 95% confidence interval for F(d) for each distance class.

Sibship structure was analysed using the software COLONY [START_REF] Wang | Estimating pairwise relatedness from dominant genetic markers[END_REF][START_REF] Wang | Parental and sibship inference from multilocus genotype data under polygamy[END_REF][START_REF] Jones | COLONY: a program for parentage and sibship inference from multilocus genotype data[END_REF]. For each pair of samples, COLONY computes the likelihood of assignment into half sibs (one parent in common), full sibs (same mother and father) or clones. We ran COLONY using the dataset containing one ramet per multilocus genotype (280 samples). In order to find the best assignment with the maximum likelihood, we chose the "Long run", "Full likelihood" and "High precision" computing options, together with no update of allele frequencies, no sibship size prior, dioecious species with male and female polygamy, and no known parental genotypes or sibships.

Clonality

Using the complete dataset, we estimated clonality as:

1 -( 𝐺-1 𝑁-1 ),
with N, the number of samples and G, the number of multilocus genotypes [START_REF] Arnaud-Haond | Standardizing methods to address clonality in population studies[END_REF]. The location and elevation (over a range of a few meters) of sampled saplings were plotted on a digital elevation model (DEM) of the Allier River using QGIS© v2.18.21 (QGIS Geographic Information System. Open Source Geospatial Foundation Project. http://qgis.org) Statistical analyses were performed using R©v3.5.1. (R Development Core Team, 2005). To test for differences in the spatial elevational and age distributions of replicated genotypes, we conducted non-parametric test (normality of the data were tested using Shapiro-Wilk). We conducted Wilcoxon test to test for differences in the distribution of elevation values of replicated and non-replicated genotypes. We conducted Fisher exact tests to test for differences in proportion of replicated genotypes in relation to their position along recruitment bands; centre of a band versus its upstream and downstream ends (i.e. the extremities of a band) and their age-class. To test for a change in density with cohort age, we compared the within-group distance between individuals using a non-parametric Dunn test of multiple comparisons. Because of unequal sample sizes among the cohorts, the test was performed on all the groups of young and old individuals and two sets of three randomly chosen groups of middle-aged individuals.

Results

Genetic diversity

Genetic diversity was high in our sample population (He = 0.859) and was comparable among the three age classes (young, He = 0.858; middle-aged, He = 0.859; old He = 0.848; Table 2). The effective number of alleles, NAe, was 10.79 (Table 1). The multilocus inbreeding coefficient was low (Fis = 0.011). Our Fis value does not show deviation from Hardy-Weinberg equilibrium (p-value = 0.240) indicating no population sub-structure. This absence of population subdivision was also confirmed by the STRUCTURE runs indicating a best number of clusters equal to 1 (data not shown).

Clonality

Out of the 348 individuals sampled we identified 280 multilocus genotypes (Table 2), of which 51 were replicated (i.e. individuals with two or more ramets) and 229 were represented by a single sample. GenALEx and COLONY (BestClone output) identified the same multilocus genotypes. Clonality was 19% for the whole population. We did not detect ramets of the same multilocus genotype in different gravel bars along the 1.6 km river reach we sampled. Clonality values were comparable among the three cohorts (Table 2) and the proportion of replicated genotypes did not differ among age cohorts (Fig. 3a, Fisher test p-value = 0.202). Elevation (i.e. topography) on the riverbank did not explain the abundance of replicated genotypes (Wilcoxon test p-value = 0.7401). However, replicated genotypes were more abundant at the upstream and downstream ends of the recruitment bands (Fig. 3b, Fisher test p-value = 1.032e-5). There were also more replicated genotypes located at the most exposed ends of the recruitment bands (relative to the water flow in blue in Fig. 4).

Spatial Genetic Structure

Along the 1.6 km river reach, no SGS could be detected when considering the whole sample population. However, we found that individuals in the first distance class (55 m) were significantly more related to one another than on average in the population (Fig. 2, F(d) significantly different from 0). Within patches (Fig. 2), we found a significant decline in the pairwise kinship coefficient with linear distance (p-value = 0.029), for the young individuals, although all F(d) values were not significantly different from 0 (within the 95 % confidence interval). No FSGS was detected for the middle-aged and old patches. COLONY identified 15 full-sibs (FS) and 44 half-sibs (HS) pairs within patches, of which 9 and 16, respectively, with a probability greater than 0.75. In particular, within one patch we found a family group of five FS within distances of two meters. The mean distance between individuals was significantly smaller in young and middle-aged patches compared to old patches (Dunn test, p-value < 0.001for both) but there was no difference between young and middle-aged patches (Dunn test, p-value = 0.1547).

Discussion

Genetic diversity and clonality

The high genetic diversity observed in the Allier River population studied (expected heterozygosity, He = 0.859 and effective number of alleles, NAe = 10.79) is consistent with literature data on the genetic diversity of trees and is explained by life history traits promoting the maintenance of high diversity within forest tree species such as efficient gene flow mechanisms, mostly outcrossing breeding systems and longevity [START_REF] Austerlitz | Effects of colonization processes on genetic diversity: differences between annual plants and tree species[END_REF]. It is also consistent with other studied P. nigra populations using SSR markers. At a regional scale (hundreds of km), the expected heterozygosity of 17 populations originating from 7 catchment systems in Europe was 0.756 (Smulders et al., 2008). A similar value (0.753) was found in Western Europe for 13 sites, although this was over an even larger spatial scale (several hundreds of km) [START_REF] Dewoody | Genetic and morphological differentiation in Populus nigra L.: isolation by colonization or isolation by adaptation?[END_REF]. At the catchment scale (tens of kilometres), genetic diversity ranged from 0.73 along the Drôme River [START_REF] Imbert | Dispersal and gene flow of Populus nigra (Salicaceae) along a dynamic river system[END_REF] to 0.79 along the Danube River [START_REF] Jelić | Indigenous forests of European black poplar along the Danube River: genetic structure and reliable detection of introgression[END_REF]. At the population scale (2-5 km), He ranged from 0.69 along the Loire River [START_REF] Chenault | SSR-based analysis of clonality, spatial genetic structure and introgression from the Lombardy poplar into a natural population of Populus nigra L. along the Loire River[END_REF] to 0.82 along the Morava River [START_REF] Pospíšková | Population structure and parentage analysis of black poplar along the Morava River[END_REF] and NAe ranged from 3.83 along the Eder River in Germany [START_REF] Rathmacher | Short-distance gene flow in Populus nigra L. accounts for small-scale spatial genetic structures: implications for in situ conservation measures[END_REF] to 8.53 across the entire Danube catchment system [START_REF] Jelić | Indigenous forests of European black poplar along the Danube River: genetic structure and reliable detection of introgression[END_REF]. The lower values reported in a number of these studies can be explained by a correspondingly high reported degree of population fragmentation, isolation and limited potential for regeneration. However, each study used different SSR marker sets, which can also explain the observed differences. Indeed, some SSR markers are more polymorphic than others. For example, in the Allier population, two SRR markers (WPMS22 and 10 PMGC2385) were highly polymorphic. Nevertheless, the high genetic diversity of our population located in a Nature Reserve where the flow regime is largely unregulated and morphodynamics are only moderately impacted by human activities [START_REF] Garófano-Gómez | Vegetation succession processes and fluvial dynamics of a mobile temperate riparian ecosystem: the lower Allier River (France). Géomorphologie: relief, processus[END_REF], highlights the importance of this population as an asset in the conservation of P. nigra genetic resources.

When considering levels of clonality, our value (19%) was comparable to the levels of clonality (14%) found in 17 populations in Central Europe (Smulders et al., 2008), mostly sampled along regulated rivers.

Regulated rivers tend to have a reduced surface area of bare alluvial bars. Thus, seed recruitment is highly restricted and the main reproductive strategy for poplar is to regenerate vegetatively. Ramets are produced by a diversity of mechanisms each with their own influence on SGS. Root-borne sucker shoots or burial of individuals mostly produce discrete units of 2-4 ramets growing close to one another (typically only over a few meters; [START_REF] Barsoum | Variations in levels of clonality among Populus nigra L. stands of different ages[END_REF] but longer distances are reported in other Populus species [START_REF] Wiehle | Root suckering patterns in Populus euphratica (Euphrates poplar, Salicaceae)[END_REF]. On the other hand, breakage during floods can spread translocated fragments downstream, transporting them over a wider range, up to several km [START_REF] Barsoum | Relative contributions of sexual and asexual regeneration strategies in Populus nigra and Salix alba during the first years of establishment on a braided gravel bed river[END_REF][START_REF] Barsoum | Variations in levels of clonality among Populus nigra L. stands of different ages[END_REF]. In France, studies based on microsatellites show levels of clonality ranging from 1% (Drôme river, [START_REF] Imbert | Dispersal and gene flow of Populus nigra (Salicaceae) along a dynamic river system[END_REF], to 49 % (lower Loire River, [START_REF] Chenault | SSR-based analysis of clonality, spatial genetic structure and introgression from the Lombardy poplar into a natural population of Populus nigra L. along the Loire River[END_REF]. Chenault et al. argue that their study site was not suitable for seedling recruitment because of anthropogenic disturbances and suggest that regeneration occurred via layering during flood disturbance events. We detected no translocated fragments along the Allier River study reach. Over a 30 km Garonne River study reach, [START_REF] Barsoum | Relative contributions of sexual and asexual regeneration strategies in Populus nigra and Salix alba during the first years of establishment on a braided gravel bed river[END_REF] found many translocated fragments, but our Allier River study reach was only 1.6 km long, which reduced the chances of encountering translocated fragments. Comparing levels of clonality with other studies must however be undertaken with caution because different sampling strategies were used, some of which were designed to avoid over-representing the number of ramets from the same MLG.

Dispersal inferred from SGS and FSGS

As our aim was to understand the local pattern of dispersal (by pollen or seeds) on genetic structure, we kept only one ramet per multilocus genotype in our dataset. No population structure was detected along the 1.6 km section studied, however, individuals located no further than 55 m from one another (first distance class used in our global SGS analysis, Fig. 2) were significantly more related than by chance. This suggests that at short distances (50 m) dispersal is not homogeneous, but that at the 1.6 km scale of the study reach, seeds from different parent trees are well mixed. In order to test the generality of our results, we reanalysed the data obtained by one of us (see the reanalysis of the [START_REF] Barsoum | Variations in levels of clonality among Populus nigra L. stands of different ages[END_REF] data in supplementary material) in another river catchment (Garonne, France). The same analyses used for this study were performed (see supplementary material)). This reanalysis confirmed the absence of population structure over short distances (up to 15 meters). We however found a significant SGS along the 30-km stretch where individuals were highly related in the first two distance classes (corresponding to distances of up to 2 km) (p value < 0.001) (Fig S2). This result indicates that gene flow is significantly reduced, but only beyond distances of 2 km.

Several studies addressed SGS among populations or patches of P. nigra over a large range of distances.

Across Europe, isolation by distance (IBD) was reported in the Danube River basin over hundreds of kilometres [START_REF] Jelić | Indigenous forests of European black poplar along the Danube River: genetic structure and reliable detection of introgression[END_REF], along a 50 km stretch of the Drôme River, France [START_REF] Imbert | Dispersal and gene flow of Populus nigra (Salicaceae) along a dynamic river system[END_REF] and even over a 2 km stretch of the Eder River, Germany [START_REF] Rathmacher | Short-distance gene flow in Populus nigra L. accounts for small-scale spatial genetic structures: implications for in situ conservation measures[END_REF]. SGS was found significant along the Morava and Eder Rivers, [START_REF] Pospíšková | Population structure and parentage analysis of black poplar along the Morava River[END_REF][START_REF] Rathmacher | Short-distance gene flow in Populus nigra L. accounts for small-scale spatial genetic structures: implications for in situ conservation measures[END_REF] at a spatial scale of tens of kilometres. This was likely due to the high degree of isolation of these populations, which were separated from other populations by distances of at least 15 km. Many studies have additionally found genetic differentiation within populations, suggesting barriers to gene flow, or partially isolated populations [START_REF] Imbert | Dispersal and gene flow of Populus nigra (Salicaceae) along a dynamic river system[END_REF][START_REF] Pospíšková | Population structure and parentage analysis of black poplar along the Morava River[END_REF][START_REF] Rathmacher | Short-distance gene flow in Populus nigra L. accounts for small-scale spatial genetic structures: implications for in situ conservation measures[END_REF]. [START_REF] Pospíšková | Population structure and parentage analysis of black poplar along the Morava River[END_REF] suggested that the occurrence of genetic spatial patterns is related to pollen dispersion dynamics based on observations of pollen dispersion ranging from 10 to 230 meters along the Morava River, Czechoslovakia. [START_REF] Braatne | Life history, ecology and reproduction of riparian cottonwoods in North America[END_REF] suggested that most seeds transported by wind are generally deposited within only a few hundred meters of the mother tree. This suggests that despite the wide distributional range of P. nigra, most pollen and seeds disperse over short distances (Fig. 5).

At the fine scale (within patches), we detected a significant FSGS for the young cohorts (around five years old) both in the Allier (Fig. 2) and in the Garonne (Fig. S2). This indicates that across short dispersal distances seeds from a single tree are not homogeneously distributed and can become entangled together to form a single aggregate during transportation by water or other means (e.g. a broken branch carrying open seed pods which fell into the river). Seeds are released surrounded by poplar fluff that often aggregate into a package while transported in the air or by water and colonize the area where it lands onshore. When these aggregates disperse, they are mixed with aggregates from other trees that are transported by the river and reach gravel bars where they germinate.

These processes create dense recruitment bands and could potentially result in the successful fine scale recruitment of P. nigra seedlings originating from a few parental trees. Indeed, both in the Allier and in the Garonne (see supplementary material), we found several pairs of FS and HS within patches, some of which very close to each other. In the Allier, we found a group of five FS from the same mother tree at distances of two meters in a young patch. Similarly, in the Garonne we found significantly more FS in young patches compared to middle-aged and old patches (See supplementary material Table S2). Thus, at the fine-scale (a few meters), neighbouring seedlings can be strongly related because the mixing of aggregate of seeds from different mother trees is imperfect.

Conversely, at the scale of a few kilometres, the mixing of seeds could lead to the absence of significant differences in genetic structure (Fig. 5b).

Intra-specific interactions

The occurrence of replicated genotypes mostly at the upstream and downstream ends of the recruitment bands along the Allier River indicates potential positive intra-specific facilitation interactions at an early stage of gravel bar colonisation. Facilitation is defined as 'an interaction in which the presence of one species alters the environment in a way that enhances the growth, survival and reproduction of a second species' [START_REF] Bronstein | The evolution of facilitation and mutualism[END_REF](Bronstein, , p 1160)), a definition which is also valid when considering different genotypes of the same species [START_REF] Mcintire | Facilitation as a ubiquitous driver of biodiversity[END_REF]. The upstream and downstream ends of the recruitment bands are more exposed to hydrodynamic forces than the centre of the recruitment bands because they are located at lower elevations and are directly exposed to high flow velocities during flooding events. This mechanical stress is known to directly enhance vegetative regeneration [START_REF] Francis | The survival and growth response of Populus nigra fragments to differing hydrogeomorphological conditions[END_REF][START_REF] Barsoum | Variations in levels of clonality among Populus nigra L. stands of different ages[END_REF]. At the same study site on the Allier River, Corenblit et al. (2016a) demonstrated that upstream individuals trap fine sediment, resulting in the formation of a sediment tail (see also [START_REF] Rodrigues | Flow and sediment dynamics in the vegetated secondary channels of an anabranching river: the Loire River (France)[END_REF][START_REF] Rodrigues | In-channel woody vegetation controls on sedimentary processes and the sedimentary record within alluvial environments: a modern example of an anabranch of the River Loire, France[END_REF][START_REF] Hortobágyi | Niche construction within riparian corridors. Part I: Exploring biogeomorphic feedback windows of three pioneer riparian species (Allier River, France)[END_REF]. These sediment tails, composed of fine sediment and organic matter, create downstream habitats that are favourable for vegetation growth [START_REF] Francis | Perspectives on biogeomorphology, ecosystem engineering and self-organisation in island-braided fluvial ecosystems[END_REF][START_REF] Corenblit | The biogeomorphological life cycle of poplars during the fluvial biogeomorphological succession: a special focus on Populus nigra L.: The biogeomorphological life cycle of poplars[END_REF]. This phenomenon was also observed along the Drôme and Garonne Rivers in France [START_REF] Barsoum | Relative contributions of sexual and asexual regeneration strategies in Populus nigra and Salix alba during the first years of establishment on a braided gravel bed river[END_REF]Corenblit et al., 2016b) and the Tagliamento River in Italy [START_REF] Gurnell | Effects of deposited wood on biocomplexity of river corridors[END_REF]. Along the Allier River, poplar recruitment bands rapidly end up in a higher elevation situation compared to the surrounding alluvial surfaces [START_REF] Hortobágyi | Niche construction within riparian corridors. Part I: Exploring biogeomorphic feedback windows of three pioneer riparian species (Allier River, France)[END_REF] through sediment accretion. Individuals at the centre of these bands may thus benefit from habitat improvement and protection from mechanical stress provided by individuals at the upstream end of these bands. By increasing stem density at the most exposed parts of recruitment bands, clonality increases sediment trapping, thereby strengthening the capacity of the downstream individuals to survive and grow. At high densities, individuals at the centre of these bands also benefit from their own local short range mutually protective effect. In such a situation, a facilitation interaction has been suggested [START_REF] Barsoum | Relative contributions of sexual and asexual regeneration strategies in Populus nigra and Salix alba during the first years of establishment on a braided gravel bed river[END_REF][START_REF] Gurnell | Effects of deposited wood on biocomplexity of river corridors[END_REF][START_REF] Corenblit | The biogeomorphological life cycle of poplars during the fluvial biogeomorphological succession: a special focus on Populus nigra L.: The biogeomorphological life cycle of poplars[END_REF]Corenblit et al., , 2016b[START_REF] Corenblit | Niche construction within riparian corridors. Part II: The unexplored role of positive intraspecific interactions in Salicaceae species[END_REF].

At the recruitment stage, aggregates of related P. nigra seeds reach the bare surface of gravel bars and produce a significant FSGS among young individuals (Fig. 5c). These seedlings can be very abundant (e.g. > 4000/m²; [START_REF] Braatne | Life history, ecology and reproduction of riparian cottonwoods in North America[END_REF] and high seedling densities in a disturbed and stressful environment allow facilitation processes to occur. In these situations, each individual benefits from the presence of others regardless of their relatedness. In riparian corridors, the habitat changes caused by poplars during the establishment phase were described as niche construction [START_REF] Corenblit | The biogeomorphological life cycle of poplars during the fluvial biogeomorphological succession: a special focus on Populus nigra L.: The biogeomorphological life cycle of poplars[END_REF][START_REF] Hortobágyi | Niche construction within riparian corridors. Part I: Exploring biogeomorphic feedback windows of three pioneer riparian species (Allier River, France)[END_REF]. This results in older poplars becoming progressively disconnected from the active channel and experiencing less frequent hydrogeomorphological disturbances such as mechanical and hydric stress (González et al., 2010;Corenblit et al., 2016a). According to the stress gradient hypothesis [START_REF] Bertness | Positive interactions in communities[END_REF], different stress levels change the type of interaction between individuals from positive (facilitation) in stressful environments to negative (competition) in benign environments. In our case, as observed for the conditionality of mutualistic interactions (Bronstein, 1994), the interaction shifts during the lifetime of a single individual. As young individuals stabilize together their habitat during establishment, intra-specific interactions shift from positive to negative (facilitation to competition) through self-thinning [START_REF] Yoda | Self-thinning in overcrowded pure stands under cultivated and natural conditions[END_REF][START_REF] White | Correlated changes in plant size and number in plant populations[END_REF] (Fig. 5c). Self-thinning tends to significantly reduce the density of trees growing very close to one another, and we showed that the average distance between individuals increases with cohort age, resulting in a lower density in older cohorts. This reduces shortdistance relatedness by removing some individuals strongly related to their neighbours. During this process, only a few individuals from the original seed aggregates remain and will eventually reach sexual maturity [START_REF] Corenblit | The biogeomorphological life cycle of poplars during the fluvial biogeomorphological succession: a special focus on Populus nigra L.: The biogeomorphological life cycle of poplars[END_REF](Corenblit et al., , 2018)). FSGS is, therefore, gradually lost as stands mature.

Studying the spatial genetic structure of populations at different spatial scales (SGS and FSGS) and for different age cohorts allowed us to reveal intra-specific interactions and how these interactions change through different life stages. [START_REF] Corenblit | Niche construction within riparian corridors. Part II: The unexplored role of positive intraspecific interactions in Salicaceae species[END_REF] suggest that niche construction processes, in addition to indirect facilitation interactions, could in particular cases include direct intraspecific interactions such as cooperation or altruism, which would involve preferential positive interactions among related individuals. Our result showing that related (but non-clonal) individuals are present in young patches is in line with this hypothesis. The validation of the hypothesis that niche construction processes involve cooperation or altruism will however require (i) to describe the FSGS at the very first stages of colonization (first-year seedlings) and (ii) experimental in situ or ex situ tests of more efficient niche construction by related compared to non-related individuals.

Concluding remarks

We chose to study a site located in a natural reserve where the flow regime is unregulated and morphodynamic processes are only moderately impacted by human activities, promoting the formation of bare gravel bars suitable for P. nigra regeneration, i.e. a site where natural processes are largely undisturbed and where P. nigra can thrive naturally. In this site, genetic diversity was high, similar to, or even higher than most data available for European rivers and we found a significant SGS for the younger cohorts compared to older cohorts were SGS was not significant.

Facilitation is known to occur through niche construction in black poplars at the early stages of recruitment when seedling density is high, and the environment is disturbed and stressful [START_REF] Corenblit | Niche construction within riparian corridors. Part II: The unexplored role of positive intraspecific interactions in Salicaceae species[END_REF].

Our results suggest that facilitation could be directed towards individuals that happen to be genetically related because seeds from a single tree are aggregated in the poplar fluff. This suggests that direct positive interactions among related individuals, such as cooperation or altruism, could take place in the niche construction process.

Clonality enhances this facilitative effect by increasing stem density at the most exposed parts of recruitment bands. Once established and stress is reduced, young poplars seem to shift to a competitive interaction.

Conservation measures should make sure that suitable sites allowing high densities of seedlings and saplings are available to ensure establishment of future mature individuals through these facilitation processes.

Finally, our results suggest that facilitation can be directed towards related individuals. This suggests that direct positive interactions among related individuals, such as cooperation or altruism, could take place in the niche construction process. Studying these intraspecific interactions could help design better future conservation strategies.

Figure legends

Tables

Table 1 Population genetic parameters of the Allier population for each locus and all loci combined. N = number of individuals; Na = total number of alleles; NAe = effective number of alleles [START_REF] Nielsen | Estimating effective paternity number in social insects and the effective number of alleles in a population[END_REF]; He and Ho = expected and observed heterozygosities, respectively; Fis = inbreeding coefficient; P-value (Fis ≠ 0) = the p-value of the permutation test. Na, NAe, He, Ho, and Fis values values were calculated using one sample per multilocus genotype (i.e. N = 280).*except the GCPM2995 marker which was removed from the analysis because of the high null allele frequency.

Locus

Fig. 1

 1 Fig. 1 Aerial photograph of the Allier River study reach, showing the position of the P. nigra saplings/trees sampled along three gravel bars. Colours represent the age of the individuals sampled; purple: old individuals; blue: middle-aged individuals and yellow: young individuals. River flow direction is from South to North.

Fig. 2

 2 Fig. 2 Spatial autocorrelation plots for the whole population, and the young, middle-aged and old cohorts separately. The average kinship coefficient F(d) is plotted against geographical distance between individuals. Confidence intervals (95%) are indicated by dashed lines. Values within the confidence interval are not significantly different from 0 (α = 0.05).

Fig. 3

 3 Fig. 3 Count of unique genotypes (black bars) and replicated genotypes (white bars) in the Allier population, as a function of (a) the age cohort (Young ≈ 5 years old; Middle-aged ≈ 10 years old; Old ≈ 20 years old) and (b) position in the recruitment bands (upstream and downstream ends are most exposed to river flow).

Fig. 4

 4 Fig. 4 Digital elevation model (DEM) of one gravel bar sampled in the Allier. White dots are unique genotypes. Red dots are replicated genotypes. The colour scale shows the elevation, from low (blue) to high (brown) values of absolute altitudes (m). Blue arrows represent preferential flow paths during high flow. Our field observations confirm these preferential flow pathways. The green lines delimit the envelopes of the recruitment bands.

Fig. 5

 5 Fig. 5 Hypothetical model illustrating SGS at the different scales of our study. At the corridor scale (a), SGS is significant because gene flow occurs mostly over a few kilometres and seed dispersal is reduced over longer distances. Along the Allier we only sampled a 1.6 km reach and we have no information for the upstream and downstream sections of our sampling reach. At the reach (b) and the regeneration band (c) scales, young individuals form patches of potentially related individuals (identified through the FSGS analysis); these protect one another at the establishment stage (facilitation). When aging, intra-specific competition takes over, leaving behind only one or a few individuals from each patch of related individuals. At this stage, FSGS is lost. For (b) and (c), the colored dots each represent a different genotype; red = genotype A, green = genotype B and blue = genotype C.

  Figure legend

Fig. S2

 S2 Fig. S2 Spatial autocorrelation plots for the whole population, the young, middle-aged and old cohorts. The average kinship coefficient F(d) is plotted against geographical distance between individuals. Confidence intervals (95%) are indicated by dashed lines. All values within the confidence interval are not significantly different from 0 (α = 0.05). All values within the confidence interval are not significantly different from 0 (α = 0.05)

Table 2

 2 Population genetic parameters of the different age cohorts sampled along the Allier (all samples of a given age cohort are combined). N, number of individuals; G, number of multilocus genotypes; Clonality; Na, total number of alleles; NAe, effective number of alleles[START_REF] Nielsen | Estimating effective paternity number in social insects and the effective number of alleles in a population[END_REF]; He and Ho, expected and observed heterozygosities, respectively; Fi inbreeding coefficient; P value (Fi𝑠 ≠ 0), p value of the permutation test. Na, NAe, He, Ho and Fis values were calculated using one ramet per multilocus genotype.

		N	Null allele frequency	Na	NAe	He	Ho	Fis		P-value (Fi ≠ 0)
	GCPM2995	280	0.137		15	5.88	0.830	0.312	0.312	0.000
	ORPM221	280	-0.010		15	8.39	0.881	0.896	-0.018	0.401
	PMGC14	280	0.004		7	3.92	0.745	0.739	0.008	0.801
	PMGC93	280	0.009		6	4.18	0.761	0.741	0.026	0.409
	PMGC2578	280	-0.007		18	6.29	0.841	0.853	-0.014	0.554
	PMGC2385	280	0.026		32	20.81	0.952	0.896	0.059	0.000
	WPMS13	280	-0.010		15	8.04	0.876	0.893	-0.020	0.372
	WPMS22	280	0.014		43	23.88	0.958	0.929	0.031	0.020
	All markers*	280	n.a.		19.43	10.79	0.859	0.850	0.011	0.179
	Cohort	Age	N	G	Clonality	Na		NAe	He	Ho	Fis	P value
		(years)										(Fi ≠ 𝟎)
	Young	≈ 5	28	23	0.19	11.57		10.08	0.858	0.894	-0.043	0.159
	Middle-aged	≈ 10	276	222	0.19	19.29		10.88	0.859	0.851	0.009	0.310
	Old	≈ 20	44	35	0.21	12.86		9.51	0.848	0.812	0.043	0.099
	All		348	280	0.19	19.43		10.79	0.859	0.850	0.011	0.179

Table S1

 S1 Genetic diversity characteristics of the different age cohorts sampled along the 745 Garonne Rivers (all samples of a given age cohort are combined). N, number of individuals; G, 746 number of multilocus genotypes; 1-((G-1)/(N-1)), clonality; Na, total number of alleles; NAe, 747 effective number of alleles (Nielsen et al. 2003); He and Ho, expected and observed 748 heterozygosities, respectively; Fi inbreeding coefficient; P value (Fi ≠ 0), p value of the 749 permutation test. Na, NAe, He, Ho, He/Ho and Fis values were calculated using the number of 750 multilocus genotypes, G. 751

	Cohort	Age (years)	N	G	1 -	(𝐺 -1) (𝑁 -1)	Na	NAe He	Ho	Fis	P value (Fi ≠ 𝟎)
	Young	5.6 (±1.4) 137 132		0.04	19.4	7.01 0.822 0.815 0.009 0.563
							0
	Middle-aged 8 (±1.6)	136 111		0.19	18.0	6.70 0.809 0.771 0.047 0.006
							0
	Old	17.6	144 123		0.15	16.4	6.33 0.801 0.761 0.050 0.002
		(±2.5)					0
	All		417 360		0.14	22.6 6.65 0.812 0.783 0.037 0.000
	752

Table S2

 S2 Repartition of Full-sibs (FS) and Half-sibs (HF) in the different age cohorts sampled 753 along the Garonne river (all samples of a given age cohort are combined). For both FS and HF 754 the chi² test was significant. 755

			Full-sibs (FS)		Half-Sibs (HS)
			Nb of
	Age class	Nb of FS	non-FS	Nb of pair in total	Nb of HS Nb of non-HS	Nb of pair in total
	Young	13	914	927	16	911	927
	Middle-aged	2	730	732	17	715	732
	Old	4	844	848	7	841	848
	Total	19	2488	2507	40	2467	2507
		FS chi²	108,3 ***	HS chi²	144,8 ***
	756
	757
	758
	759
	760
	761
	27

Acknowledgements

The Garonne work was funded under the EU research contract: EVK1-CT-1999-00031FLOBAR2 (Floodplain Biodiversity and Restoration).

The first author was funded for his PhD by the French Ministry of National Education, Higher Education and Research. This research was supported by the French government IDEX-ISITE initiative 16-IDEX-0001 (CAP 20-25). Financial support was provided by the LTSER Zone Atelier Loire -CNRS, the Fédération des Recherches en Environnement (FR 3467 UCA/CNRS/INRA), Clermont-Ferrand and I-SITE Clermont CAP 20-25 projet Emergence 2018. have no conflict of interest.

The genetics data (tables of genotypes) and R code used in this study are currently being submitted to Zenodo open-access repository (https://about.zenodo.org/).

Code availability: not applicable

Supplementary material

M&M

The data used in [START_REF] Barsoum | Variations in levels of clonality among Populus nigra L. stands of different ages[END_REF] to study clonality along a 30 km reach of the Garonne river was analysed to study the FSGS. We provide here the general information regarding sampling and genotyping. For further details see [START_REF] Barsoum | Variations in levels of clonality among Populus nigra L. stands of different ages[END_REF].

Sampling strategy along the Garonne River

P. nigra stands were sampled in 2004 along a 30 km study reach. Saplings or trees were categorised using the same three age categories described for the Allier River (i.e. young, middle-aged, old). For each age category, ten sampling locations (patches) were selected. At each of these sampling locations 15 saplings or trees were selected by first selecting one individual and then its 14 downstream nearest neighbours, resulting in a total of 450 individuals sampled. In order to avoid sampling re-sprouting stems of the same individual, the stem with the greatest diameter in a patch was sampled. In each sampling location, the first two trees sampled were used as spatial references and all the other trees were localized using their distance to these two trees. Using these distances, only the 417 trees that could be mapped precisely were kept for further analyses.

DNA extraction, genotyping and genetic analysis for the Garonne

Five microsatellite markers WPMS03, WPMS05, WPMS07, WPMS09 and WPMS12 (Smulders et al. 2003) were used in the analyses. To remove the impact of clonality on genetic structure, further analyses were performed with only one individual of each replicated genotype per sampling location resulting in a dataset of 360 individuals. We used the same procedures as in the Allier to study SGS and FSGS.

Results

Genetic diversity was high in the Garonne population (He = 0.812) and clonality was 14% (Table S1). We found a strong SGS along the 30 km section of the Garonne (Fig. S1). For the analysis over the whole 30 km reach (SGS), individuals were highly related in the first two distance classes (corresponding to distances of up to 2 km) (p value < 0.001).We found a significant spatial autocorrelation (FSGS) among the young individuals along the Garonne River (p-value = 0.041). No FSGS was detected for the middle-aged and old individuals sampled (Fig. S1). Within patches, COLONY identified 19 full-sibs and 95 half-sib pairs with a probability greater than 0.7 and 0.5 respectively. In particular, the highest number of full-sibs pairs were found in the youngest cohort (Table S2).