Lydia Bousset

P-Y Henry

P Sourrouille

P Jarne

Henry Py

Population biology of the invasive freshwater snail Physa

Keywords: demography, ecological characterization, genetic structure, microsatellites, snail

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Introduction

Natural populations are influenced by both demographic and evolutionary factors. Although no unified framework has been proposed up to now to consider both groups of factors at once, it remains possible to adopt a pluralistic approach, that is to combine results from the analysis of population genetic structure (see, e.g. [START_REF] Slatkin | Gene flow in natural populations[END_REF][START_REF] Hartl | Principles of Population Genetics[END_REF][START_REF] Rousset | Inferences from spatial population genetics[END_REF] with those from population demography (see, e.g. [START_REF] Ebert | Plant and Animal Populations[END_REF][START_REF] Caswell | Matrix Population Models[END_REF]. Further information can be gathered from environmental characterization of habitats or ecological approaches. In the simplest version of the pluralistic approach, even a simple characterization of habitats may help in interpreting genetic patterns. [START_REF] Giles | Genetic differentiation in Silene dioica metapopulations: estimation of spatiotemporal effects in a successional plant species[END_REF] showed that population age explains the genetic differentiation of populations in a plant, as expected from metapopulation theory. Habitat openness predicts the amount of genetic variability maintained within populations of a freshwater snail (Charbonnel et al. 2002a). A more sophisticated approach includes the analysis of the same factor with different methods. The most obvious example is migration: it might be inferred under specific hypotheses from the spatial distribution of genetic variability (see, e.g. [START_REF] Hartl | Principles of Population Genetics[END_REF], but also from cohort analysis [START_REF] Ebert | Plant and Animal Populations[END_REF] or capture-mark-recapture methodology [START_REF] Schwarz | Estimating animal abundance: review III[END_REF]. This has been done in a few cases only and has sometimes provided surprisingly congruent estimates [START_REF] Johnson | Neighbourhood size and the importance of barriers to gene flow in an intertidal snail[END_REF]review in Ims & Yoccoz 1997;[START_REF] Rousset | Inferences from spatial population genetics[END_REF]. However, the pluralistic approach remains underrepresented in the literature (see References in Charbonnel et al. 2002c). We here tried to follow such an approach, focusing on four influent factors in population biology, namely mating systems, population size, demography (bottlenecks) and migration.

Comment citer ce document : Bousset, L., Henry, P.-Y., Sourrouille, P., Jarne, P (2004). Population biology of the invasive freshwater snail Physa acuta approached through genetic markers, ecological characterization and demography. Molecular Ecology, 13 (7), 2023-36. , DOI : 10.1111/j. 1365-294X.2004.02200.x This file is the post-print of: Bousset et al. Molecular Ecology (2004) 13:2023-2036doi: 10.1111/j.1365-294X.2004.02200.x 2

In this perspective, hermaphroditic freshwater snails (Gastropoda: Pulmonata) might constitute appropriate biological models [START_REF] Brown | Freshwater Snails of Africa and Their Medical Importance[END_REF][START_REF] Städler | Population biology, genetic structure, and mating system parameters in freshwater snails[END_REF][START_REF] Dillon | The Ecology of Freshwater Molluscs[END_REF]. We focus here on Physa acuta, also referred to as P. heterostropha or P. integra [START_REF] Dillon | Populations of the European freshwater pulmonate Physa acuta are not reproductively isolated from American Physa heterostropha or Physa integra[END_REF]. This preferentially outcrossing species selffertilizes at population rates ranging from 0.1 to 0.3 [START_REF] Wethington | Selfing, outcrossing, and mixed mating in the freshwater snail Physa heterostropha: lifetime fitness and inbreeding depression[END_REF][START_REF] Jarne | The influence of self-fertilization and grouping on fitness attributes in the freshwater snail Physa acuta: population and individual inbreeding depression[END_REF][START_REF] Tsitrone | Delayed selfing and resource reallocations in relation to mate availability in the freshwater snail Physa acuta[END_REF]Henry et al. submitted for publication). P. acuta occupies a variety of natural and artificial habitats which both are discontinuously distributed and exhibit wide temporal variation in water availability. Habitat openness (e.g. rivers vs. ponds) and water regime (e.g. permanent vs. temporary) can therefore be used to characterize populations. This environmental variation should be associated with marked variation in population census size (see [START_REF] Brown | Freshwater Snails of Africa and Their Medical Importance[END_REF][START_REF] Dillon | The Ecology of Freshwater Molluscs[END_REF]) and therefore genetic drift, and potentially with variation in migration and extinction/recolonization processes (Charbonnel et al. 2002b,c;[START_REF] Chlyeh | Population genetics and dynamics at short spatial scale of Bulinus truncatus, the intermediate host of Schistosoma haematobium, in Morocco[END_REF]. P. acuta is an invasive species originating from North America which has widely enlarged its distribution area over the last two centuries. It is now found on all continents, and is indeed the most cosmopolitan freshwater snail [START_REF] Dillon | Populations of the European freshwater pulmonate Physa acuta are not reproductively isolated from American Physa heterostropha or Physa integra[END_REF]. This extension might have been associated with population bottlenecks during founding events, as detected in other snail species [START_REF] Meunier | Lack of molluscan host diversity and the transmission of an emerging parasitic disease in Bolivia[END_REF]Charbonnel et al. 2002a). However, the demography of this species is hardly known [START_REF] Brown | The adaptive demography of four freshwater pulmonate snails[END_REF], and the distribution of genetic variability among populations has been documented only once [START_REF] Dillon | The biogeography of sea islands: clues from the population genetics of the freshwater snail, Physa heterostropha[END_REF]. The goal of our study was to characterize the influence of the four factors mentioned above on P. acuta population biology, as part of a wider study on the demographic and evolutionary processes influencing mating system evolution [START_REF] Jarne | The influence of self-fertilization and grouping on fitness attributes in the freshwater snail Physa acuta: population and individual inbreeding depression[END_REF][START_REF] Henry | Environment-dependent inbreeding depression in a hermaphroditic freshwater snail[END_REF][START_REF] Tsitrone | Delayed selfing and resource reallocations in relation to mate availability in the freshwater snail Physa acuta[END_REF]. Genetic (microsatellite markers) and demographic and ecological data were used to interpret population patterns in the light of external data (e.g. habitat characterization) and compare estimates of population parameters (e.g. bottlenecks) derived from different sources. In total, the study included 25 populations from France. More specifically, the role of the four factors mentioned above was approached as follows:

1. The mating system was studied using a double approach. Estimates of selfing rates at the population level were obtained indirectly from the genetic survey of all populations. In a subset of populations, the selfing rate was also derived from progeny-arrays analyses, which returns individual estimates [START_REF] Ritland | Extensions of models for the estimation of mating systems using n independent loci[END_REF]. At the population level, inbreeding is a source of deviation from Hardy-Weinberg equilibrium (HWE). However, other processes (e.g. population structure) can generate such deviations (see, e.g. [START_REF] Zouros | Possible explanations of heterozygote deficiency in bivalve molluscs[END_REF]. Mean individual estimates, therefore, help in the interpretation of population estimates. 2 Populations were characterized based on habitat openness and water regime. This allowed predictions on the amount of genetic variation within populations, i.e. evaluation of the influence of genetic drift. In 11 populations around Montpellier, the demography was monitored through monthly surveys of population density and individual size distributions (see [START_REF] Brown | Freshwater Snails of Africa and Their Medical Importance[END_REF][START_REF] Ebert | Plant and Animal Populations[END_REF]) over a 2-year period. Again, this allowed predictions of density with regard to openness and water regime. 3 The occurrence of bottlenecks was analysed based both on tests of population genetic structure and on the demographic survey. Predictions were made on both genetic and demographic variation with regard to environmental descriptors. Although the genetic signals detected through genetic analyses are unlikely to take place on the same time scale as current demography (see [START_REF] Cornuet | Description and power analysis of two tests for detecting recent population bottlenecks from allele frequency data[END_REF], it is interesting to evaluate whether the current demography is indicative of trends on a longer time scale. 4 Migration was indirectly evaluated using genetic markers, for example, testing for isolation-by-distance, and contrasted among populations with regard to environmental descriptors. Migration events were also analysed based on both sudden changes in size distribution and density, and temporal variation in allelic frequencies in eight populations that were sampled twice over time. As actual demographic estimates of migration were not derived, a direct comparison with genetic estimates was not possible (see Rousset 2001 for a review). However, our approach allows detection of massive migration events and this can be contrasted among populations and compared with genetic divergence.

Comment citer ce document : Bousset, L., Henry, P.-Y., Sourrouille, P., Jarne, P (2004). Population biology of the invasive freshwater snail Physa acuta approached through genetic markers, ecological characterization and demography. Molecular Ecology, 13 (7), 2023-36. , DOI : 10.1111/j. 1365-294X.2004.02200.x This file is the post-print of: Bousset et al. Molecular Ecology (2004) 13:2023-2036doi: 10.1111/j.1365-294X.2004.02200.x 3

Materials and methods

Populations sampled, demographic survey and ecological characterization

The location of the 25 populations studied is given in Table 1. A subsample of populations located near Montpellier was more thoroughly studied, and will be referred to as the local scale. Three hundred and twentynine individuals were sampled from 11 sites both in June (t1) and October (t2) 2000 (Table 1). Unless specified, t1 and t2 samples from a given population were analysed separately. These sites were separated by distances of 0.5-19 km (Fig. 1). They were surveyed monthly from July 2000 to July 2002 for demographic and ecological purposes [START_REF] Henry | Dynamique des populations et évolution du système de reproduction. Etude chez le gastéropode Physa Acuta[END_REF]. On average, 32 (SE = 23) individuals were measured per visit and per site; size being the distance from the apex to the base of the spire (to the nearest 0.1 mm). Individuals were also marked with a paint dot to quantify survival, recruitment and migration based on the capture-mark-recapture methodology [START_REF] Schwarz | Estimating animal abundance: review III[END_REF]. However, the recapture probability was too low and population size too high to obtain meaningful results (not shown; [START_REF] Henry | Dynamique des populations et évolution du système de reproduction. Etude chez le gastéropode Physa Acuta[END_REF]. Population density was visually scored (0, 1, 5, 10, 25, 50, 100 ind/m). Note that this is significantly correlated with a more classical measure of density, the number of individuals collected per minute (Spearman rank order correlation coefficient, rs = 0.827, N = 229, P < 10 -4 ). Sites were classified according to hydrological conditions (Table 1) as temporary (drying out on at least one visit during the 2-year monthly survey) or permanent. Ponds were also distinguished from rivers. In rivers, the connectivity of successive water pools was noted (connected or disconnected). More details are given in [START_REF] Henry | Dynamique des populations et évolution du système de reproduction. Etude chez le gastéropode Physa Acuta[END_REF].

Table 1. Information on the 25 populations of Physa acuta studied (see also Fig. 1). Lez5 was considered in the study of allelic segregation only. The first 12 populations (local scale) in the table were followed for demographic purposes (see text for details)

Date is the date of sampling.indicates that snails were not detected or absent. In the 'habitat' column, Po and R stand for ponds and rivers, respectively (FC was sampled in a dam lake), and P, T and nc for permanent, temporary and not characterized, respectively.

Comment citer ce document : Bousset, L., Henry, P.-Y., Sourrouille, P., Jarne, P (2004). Population biology of the invasive freshwater snail Physa acuta approached through genetic markers, ecological characterization and demography. Molecular Ecology, 13 (7), 2023-36. , DOI : 10.1111/j. 1365-294X.2004.02200.x This file is the post-print of: Bousset et al. Molecular Ecology (2004) 13:2023-2036doi: 10.1111/j.1365-294X.2004.02200.x 4 Figure 1 Location of the 13 French populations sampled outside the Montpellier area. The populations sampled near Montpellier are indicated in the enlarged panel. Information on openness and water availability is given in Table 1.

The population genetic structure was also analysed on a global scale, using an additional sample of 13 populations (157 individuals; Table 1). Three are located around Montpellier and 10 elsewhere in France (Fig. 1). These 13 populations were either ponds or rivers (Table 1). Prior to genetic analysis, all individuals were stored in 90° alcohol or frozen.

Generating genetic data

Prior to the analysis of population structure, allelic segregation was checked at 10 microsatellite loci [START_REF] Monsutti | Dinucleotide microsatellite loci reveal a high selfing rate in the freshwater snail Physa acuta[END_REF][START_REF] Sourrouille | Microsatellite variation in the freshwater snail Physa acuta[END_REF]. No significant deviation from Mendelian expectations was observed (see online electronic supplementary material, Web Table 1). DNA extraction and characterization of variability were performed as described in [START_REF] Sourrouille | Microsatellite variation in the freshwater snail Physa acuta[END_REF]. The distribution of genetic variability in natural populations was analysed using 8 of these 10 loci, i.e. excluding Pac14 (limited variation) and AF108761 (some ambiguous scoring at glocal scale). Polymerase chain reaction (PCR) co-amplification was possible for some groups of loci (Pac1, Pac2 and Pac5; Pac4 and Pac7; AF108762 and AF108764). PCR products were run in two groups (Pac1, Pac2, Pac5, Pac4, Pac7, AF108762 and AF108764; AF108758) on an ABI Prism 310 genetic analyser. Some null homozygotes were detected, but they were concentrated at two loci (Pac4 and Pac7). The results presented here were obtained without these two loci. However, including Pac4 and Pac7 influenced our results to a limited extent (see online electronic supplementary material). Genetic markers were also used to estimate the population selfing rate (averaged over families) using progenyarrays [START_REF] Ritland | Extensions of models for the estimation of mating systems using n independent loci[END_REF] and seven of the eight loci mentioned above. The study was conducted at local scale (11 populations), and 6-13 families were studied per population. On average 6.7 (SE = 1.0) progenies were studied per family. The methodology and results are fully detailed in [START_REF] Henry | Dynamique des populations et évolution du système de reproduction. Etude chez le gastéropode Physa Acuta[END_REF].

Analysing the mating system

The selfing rate was first estimated based on population structure. Deviation from the genotypic proportions expected at HWE at each locus was tested in each population using exact tests (Rousset & Raymond 1995), and departure from HWE over all loci was evaluated using Fisher's method for combining probabilities. The unbiased estimator f ̂ of Wright's FIS was calculated according to [START_REF] Weir | Estimating F-statistics for the analysis of population structure[END_REF] using genepop 3.1 (Raymond & Rousset 1995). Selfing rates were computed as ŝ = 2f ̂/(1 + f ̂) [START_REF] Pollak | On the theory of partially inbreeding finite populations. I. Partial selfing[END_REF]. This assumes that selfing is the sole source of deviation from HWE. Population selfing rates derived from progeny-arrays were estimated using the maximum likelihood procedures implemented in mltr 2.4 [START_REF] Ritland | Extensions of models for the estimation of mating systems using n independent loci[END_REF].

Comment citer ce document : Bousset, L., Henry, P.-Y., Sourrouille, P., Jarne, P (2004). Population biology of the invasive freshwater snail Physa acuta approached through genetic markers, ecological characterization and demography. Molecular Ecology, 13 (7), 2023-36. , DOI : 10.1111/j. 1365-294X.2004.02200.x This file is the post-print of: Bousset et al. Molecular Ecology (2004) 13:2023-2036doi: 10.1111/j.1365-294X.2004.02200.x 5 Genetic diversity within populations and population size The genetic variability was described using the mean number of alleles (Nall), mean observed heterozygosity (HO) and mean gene diversity (HE) per population over all loci [START_REF] Nei | Molecular Evolutionary Genetics[END_REF]. Estimates of allelic frequencies per locus and population are given as online electronic supplementary material (Web Table 2). Deviations from genotypic equilibrium between pairs of loci were tested in each population using exact tests (Rousset & Raymond 1995). Predictions were made on genetic variation within populations, based on habitat openness and water regime (Table 2; see also Viard et al. 1997b,c;Charbonnel et al. 2002a,b,c). (i) Migration and colonization should maintain larger effective population size in rivers than in ponds; and (ii) genetic drift and extinction should be more common in temporary than in permanent habitats. More variation was therefore expected in rivers than in ponds and in permanent than in temporary habitats (Table 2). The harmonic mean density over sampling events was estimated in each population from the demographic survey, and higher values were expected in permanent than in temporary habitats (Table 2). No specific predictions were derived with regard to openness, because potentially stronger demographic bottlenecks in ponds during droughts might be counter-balanced by low density after flooding in rivers. We also tested whether current density is related to longer term population size, and could therefore predict the amount of genetic variation within populations.

Table 2. Predictions on the genetic structure (within-population polymorphism, occurrence of bottlenecks, genetic differentiation among populations, isolation-by-distance and temporal variation) and on demography based on the ecological characterization of habitats. Predictions on the genetic structure are based on the idea that (i) migration and colonization maintain larger effective population size in rivers (R) than in ponds (Po); (ii) genetic drift and extinction are more common in temporary (T) than in permanent (P) habitats

> and <, higher and lower values, respectively. =, we have no specific reason to expect differences. * Sampling drift only is considered, because no migration occurred between the two sampling events. † No specific expectation

Population demography: bottlenecks

The demographic history of populations was analysed using the method of [START_REF] Cornuet | Description and power analysis of two tests for detecting recent population bottlenecks from allele frequency data[END_REF], which is based on the heterozygosity expected in a population which size has suddenly varied. This does not require HWE. Observed heterozygosity is compared with the heterozygosity expected under mutation-drift equilibrium given the number of alleles present in the population. An excess of heterozygosity is generally expected after a bottleneck, whereas a deficit is expected in growing populations. We performed the Wilcoxon signed ranks test implemented in bottleneck [START_REF] Piry | bottleneck: a computer program for detecting recent reductions in the effective population size using allele frequency data[END_REF] in each population under the three available models of mutation (infinite allele (IAM), two-phase (TPM) and stepwise mutation (SMM) models), because there is no consensus on which model is the most appropriate for microsatellites. The occurrence of bottlenecks was also searched for in the demographic data. The three parameters used were the harmonic mean density (see above), the occurrence of low-density events (when one individual per m 2 or less was observed)

Comment citer ce document : Bousset, L., Henry, P.-Y., Sourrouille, P., Jarne, P (2004). Population biology of the invasive freshwater snail Physa acuta approached through genetic markers, ecological characterization and demography. Molecular Ecology, 13 (7), 2023-36. , DOI : 10.1111/j. 1365-294X.2004.02200.x This file is the post-print of: Bousset et al. Molecular Ecology (2004) 13:2023-2036doi: 10.1111/j.1365-294X.2004.02200.x 6 and the probability of extinction which are expected to be low, high and high, respectively, when bottlenecks are frequent. Predictions with regard to environmental descriptors on both the genetic and demographic approaches are given in Table 2. The genetic approach detects bottlenecks at a much larger time scale (of the order of Ne generations, with Ne the effective population size; [START_REF] Cornuet | Description and power analysis of two tests for detecting recent population bottlenecks from allele frequency data[END_REF] than the two years of our demographic survey. However, we reasoned that current bottlenecks in specific environments (e.g. ponds) might be indicative of historical bottlenecks.

The temporal variation in population size was further studied based on temporal genetic variation. Temporal variation of neutral genetic patterns across generations in a given population may result from stochastic processes (sampling and genetic drift) or migration (see, e.g. [START_REF] Waples | Temporal changes in allele frequencies: testing the right hypothesis[END_REF]Charbonnel et al. 2002c). In our study, variation between t1 and t2 can be interpreted in terms of stochastic sampling of individuals only. Migration can be ruled out because water pools in rivers remained disconnected between t1 and t2. Analysis of the size distributions also suggests that the individuals collected at t1 and t2 (that is before and after summer drought) belonged to the same spring cohort. Indeed, mature snails were collected at t1 at a mean size of 8.7 ± 1.6 mm over all populations, and the increase in size by t2 ranged from 4.0 to 8.1 mm per population. Massive recruitment between the two sampling dates would have resulted in a bimodal size structure and a decrease in mean size. Population temporal differentiation was tested using homogeneity tests [START_REF] Goudet | Testing differentiation in diploid populations[END_REF]. The estimator θ̂ (θ-temporal) of FST [START_REF] Weir | Estimating F-statistics for the analysis of population structure[END_REF] was calculated over all loci in each population.

Migration

The estimator θôf FST [START_REF] Weir | Estimating F-statistics for the analysis of population structure[END_REF] was calculated over all loci, both over all populations and all pairs of populations (Web Table 3) using genepop 3.1. The estimates of pairwise spatial differentiation (θspatial) were compared with θ-temporal (see above) using Mann-Whitney U-test. Less temporal than spatial variation was expected given the short time scale and the fact that sampling drift only was acting. Isolationby-distance was tested using the method of [START_REF] Rousset | Genetic differentiation and estimation of gene flow from F-statistics under isolation-bydistance[END_REF], under which a correlation is expected between the logarithm (in a two-dimensional habitat) of the geographical distance and FST/(1 -FST). This correlation was tested using matrix correlation methods based on Mantel test. A reduced major axis (RMA) regression was performed (see [START_REF] Sokal | Biometry[END_REF][START_REF] Bohonak | ibd (Isolation By Distance): a program for analyses of isolation by distance[END_REF]. Estimates of Dσ 2 were calculated from the slope of the regression, and confidence intervals on the slope were calculated by bootstrapping using ibd 1.2 [START_REF] Bohonak | ibd (Isolation By Distance): a program for analyses of isolation by distance[END_REF]. The geographical distance between two sampling points was computed as a linear distance (see Viard et al. 1997a). Predictions were produced with regard to environmental descriptors. More differentiation and isolation-by-distance was expected in ponds than in rivers, because of the expected difference in effective population size, and in temporary than in permanent habitats. Migration among populations was also approached from genetic data using assignment and exclusion methods [START_REF] Cornuet | New methods employing multilocus genotypes to select or exclude populations as origin of individuals[END_REF]. The main results, consistent with those of the FST-based analysis, are reported as online electronic supplementary material.

Immigration was analysed based on size distributions. From the distribution of individual size classes, it is possible to infer the number of cohorts occurring in a population at a given time [START_REF] Ebert | Plant and Animal Populations[END_REF]. The temporal variation of this distribution allows inferring demographic events such as recruitment of new cohorts. However, such histograms are often hard to interpret when size classes overlap over cohorts and sample size is limited. [START_REF] Persat | Typologie de distributions en classes de taille: intérêt dans l'étude des populations de poissons et d'invertébrés[END_REF] developed a method based on correspondence analysis aimed at smoothing such distributions. This method is explained fully in [START_REF] Charbonnel | De la génétique à la dynamique des populations. L'exemple du gastéropode des eaux douces Biomphalaria pfeifferi à Madagascar[END_REF] and was used in another snail species (Charbonnel et al. 2002c). In our study, immigration was expected to translate into the sudden appearance of modes that cannot be explained by growth or recruitment. This assumes that immigrants and residents had different sizes. Furthermore, the method can only detect massive immigration events. This is unlikely in ponds, because potential vectors cannot move a full cohort of subadult to adult snails, and data from closed habitats were not analysed. Immigration events were expected to be more frequent in temporary than in permanent habitats. Although this approach detects very specific events of migration, their occurrence might be used to interpret genetic patterns.

All predictions presented in Table 2 were tested using nonparametric tests [START_REF] Siegel | Nonparametric Statistics for the Behavioural Sciences[END_REF]. Note also that the comparisons of genetic and demographic patterns with regard to water regime are reported as online electronic supplementary material only (Web Table 5), as no test was significant.

Comment citer ce document : Bousset, L., Henry, P.-Y., Sourrouille, P., Jarne, P (2004). Population biology of the invasive freshwater snail Physa acuta approached through genetic markers, ecological characterization and demography. Molecular Ecology, 13 (7), 2023-36. , DOI : 10.1111/j.1365-294X.2004.02200.x

This file is the post-print of: Bousset et al. Molecular Ecology (2004) 13:2023-2036doi: 10.1111/j.1365-294X.2004.02200.x 7

Results

Analysing the mating system Fifteen of 32 multilocus tests of Hardy-Weinberg equilibrium were significant with low P-values (Table 3). Heterozygote deficiencies were detected as often in populations around Montpellier as in other populations (Table 3). The estimates of S corresponding to non-null estimates of FIS ranged from 0.12 to 0.61 (15 populations; Table 3). At the local scale, no selfing was detected in six populations (FIS not significantly different from zero). In two populations, some selfing was detected at either t1, or t2. Using progeny-arrays showed that selfing rates significantly differed from zero in three of the populations studied at local scale only. Both approaches indicated no selfing in six populations, partial selfing in two populations and conflicting results for the last three populations. The distributions were not correlated (Spearman rank order correlation coefficient, rs=-0.065, P= 0.92), although the mean values were not very different (0.06 and 0.07).

Table 3. Variation at six microsatellite loci in the 24 populations sampled. Date refers to the date of sampling when populations were sampled twice. N is the sample size and Np the number of polymorphic loci. Nall, HO and HE are, respectively, the mean number of alleles, the mean observed heterozygosity and Nei's unbiased gene diversity over all loci. Their standard deviations are indicated into parentheses. P is the probability associated with exact tests (heterozygote deficiency) of Hardy-Weinberg proportions. f ̂ and ŝ (FIS) are the estimates of Wright's FIS and the selfing rate, respectively. S was not estimated for negative values of f ̂, or in populations at Hardy-Weinberg equilibrium ŝ (PA), selfing rate estimated from progeny-arrays (at local scale only)

* Estimate differs from 0 (P < 0.05) based on bootstrap analysis.

Comment citer ce document : Bousset, L., Henry, P.-Y., Sourrouille, P., Jarne, P (2004). Population biology of the invasive freshwater snail Physa acuta approached through genetic markers, ecological characterization and demography. Molecular Ecology, 13 (7), 2023-36. , DOI : 10.1111/j.1365-294X.2004.02200.x

This file is the post-print of: Bousset et al. Molecular Ecology (2004) 13:2023-2036doi: 10.1111/j.1365-294X.2004.02200.x 8 Genetic diversity within populations and population size None of the tests of genotypic disequilibrium over all populations were significant. The genetic variation within populations is reported in Table 3 andWeb Table 2. The mean number of alleles ranged from 1.8 to 4.2, and the average gene diversity from 0.22 to 0.59 (Table 3). The number of alleles and gene diversity were higher in rivers than in ponds both at local and global scale. However, no difference was observed when comparing harmonic mean densities of individuals (Table 4). 3) between ponds and rivers. Values are given as means (SD). The 'test' column indicates either Mann-Whitney U-test (n1; n2; U) or Fisher's exact test F (n1; n2), and P is the associated probability. 'nt' holds for no test, either when testing was irrelevant (Mantel and Dσ 2 ) or not possible (no demographic data at global scale, not enough data for bottlenecks and extinctions). >, < and = are followed by #when the predictions of Table 2 were fulfilled.

Table 4. Comparisons of the genetic and demographic parameters (defined in Table

Values in the 'bottlenecks' row indicate the number of significant tests under the infinite allele model (IAM), two-phase model (TPM) and stepwise mutation model (SMM), and the total number of tests performed. km in the 'differentiation' row gives the mean geographical distance (standard deviation) between population pairs. Slope (a) values in the isolation-by-distance row are provided with their 95% confidence intervals generated by bootstrapping Population demography: bottlenecks

The null hypothesis of mutation-drift equilibrium was not rejected in most populations, whatever the mutation model (Web Table 6). One heterozygosity deficit was detected under SMM, which may indicate expansion or bottleneck [START_REF] Cornuet | Description and power analysis of two tests for detecting recent population bottlenecks from allele frequency data[END_REF]. Heterozygosity excesses (indicating bottlenecks) were detected in five populations only, and the test was significant for the three models in Mos1 and Lam1 only.

Estimates derived from the demographic survey are reported in Table 4. Low-density events were more frequent in rivers than in ponds (Table 4). Three extinction events were detected. All occurred in Lam1 (October 2000 to May 2001, October to November 2001, January to April 2002; Table 4), a site in which a strong genetic signal of bottleneck was also detected. No extinction event was observed in ponds despite five summer drying-out events.

The pairwise estimates of temporal FST ranged from 0.00 to 0.04 (Fig. 3). As expected, there was no more differentiation in open than in closed habitats (Table 4).

Comment citer ce document : Bousset, L., Henry, P.-Y., Sourrouille, P., Jarne, P (2004). Population biology of the invasive freshwater snail Physa acuta approached through genetic markers, ecological characterization and demography. Molecular Ecology, 13 (7), 2023-36. , DOI : 10.1111/j. 1365-294X.2004.02200.x This file is the post-print of: Bousset et al. Molecular Ecology (2004) 13:2023-2036doi: 10.1111/j.1365-294X.2004.02200.x 9

Figure 3 Estimates of temporal differentiation in populations sampled twice (θ-temporal; eight points) in the Montpellier area (local scale), and estimates of spatial differentiation between pairs of populations in rivers and ponds. Spatial differentiation is indicated both for the local scale (θ-spatial rivers, 11 points; θ-spatial ponds, 30 points) and outside the local scale (θ-spatial rivers, 45 points; θ-spatial ponds, 10 points). Migration

The multilocus estimates of pairwise FST are reported in Web Table 3. Spatial estimates were higher than temporal estimates, both in rivers (Mann-Whitney U = 5.0, n1 = 8, n2 = 10, P = 3 × 10 -4 ) and in ponds (U = 0.0, n1 = 8, n2 = 25, P = 4 × 10 -6 ). At a local scale, isolation-by-distance was detected when considering all populations (Mantel test, P = 0.01; note the 95% CI: -0.34 to 0.35), as well as among both ponds and rivers (Fig. 2). At global scale, isolation-by-distance was significant in both ponds (P = 0.03) and rivers (P = 10 -4 ). At both scales, more differentiation was observed in ponds than in rivers (Fig. 3), and the patterns of isolationby-distance were significantly different (no overlap of slopes; Table 4).

Size distributions were poorly informative because of the frequent occurrence of adverse climatic events (inducing high water level or turbidity) and low population densities (and thus small sample size). As immigration could be documented only if >10 individuals are sampled in successive visits, this limited our Comment citer ce document : Bousset, L., Henry, P.-Y., Sourrouille, P., Jarne, P (2004). Population biology of the invasive freshwater snail Physa acuta approached through genetic markers, ecological characterization and demography. Molecular Ecology, 13 (7), 2023-36. , DOI : 10.1111/j. 1365-294X.2004.02200.x This file is the post-print of: Bousset et al. Molecular Ecology (2004) 13:2023-2036doi: 10.1111/j.1365-294X.2004.02200.x 10 dataset to 42 visits (of 132). Although the evolution of modes was hardly identifiable, four massive immigration events were detected, and all occurred in temporary rivers (Lam1 and Lez5).

Discussion

For the sake of clarity, the four factors studied (mating systems, population size, bottlenecks and migration) are discussed separately. For each factor, we discuss what has been gained from each approach, trying to provide an integrated view at the end of each subsection. The last subsection evaluates how genetic, ecological and demographic approaches might be integrated when studying population biology.

Mating system

The hypothesis of HWE was rejected in most populations tested (Table 3). As Physa acuta is a hermaphroditic species, these results may be explained by partial selfing [START_REF] Dillon | The biogeography of sea islands: clues from the population genetics of the freshwater snail, Physa heterostropha[END_REF][START_REF] Jarne | The influence of self-fertilization and grouping on fitness attributes in the freshwater snail Physa acuta: population and individual inbreeding depression[END_REF]. Assuming that heterozygote deficiencies are indeed due to selfing, selfing rates ranged between 0.12 and 0.61 in populations departing from HWE. Lower values were obtained at local scale. This confirms that P. acuta practises both outcrossing and selfing, although [START_REF] Jarne | The influence of self-fertilization and grouping on fitness attributes in the freshwater snail Physa acuta: population and individual inbreeding depression[END_REF] reported lower values. Several forces may generate heterozygote deficiencies and it is worth comparing these values with more direct estimates derived from progeny-arrays. At the local scale, there was a slight difference only in the mean selfing rate over populations when comparing results from progeny-arrays (0.06) and population structure (0.07). A similar difference has been reported in the other highly selfing freshwater snails studied (Viard et al. 1997a;[START_REF] Charbonnel | De la génétique à la dynamique des populations. L'exemple du gastéropode des eaux douces Biomphalaria pfeifferi à Madagascar[END_REF]Trouvéet al. 2003). However, estimates from the two methods were congruent (either no or partial selfing) in 8 of the 11 populations studied.

Possible explanations for the difference between the two methods include temporal variation in the selfing rate, population substructure (Wahlund effect), biparental inbreeding and technical problems (miscoring and null alleles). We have no information on temporal variation in selfing rate. Some population differentiation may be detected even at short distances within the same river (see below), and the role of Wahlund effect remains possible. The progeny-array analysis indeed revealed some biparental inbreeding at local scale. Surprisingly, this occurred in small (size < 10 m) isolated ponds [START_REF] Henry | Dynamique des populations et évolution du système de reproduction. Etude chez le gastéropode Physa Acuta[END_REF], suggesting population structure at a very small spatial scale. However, this was in populations with selfing rates not significantly different from zero. Some miscoring was detected in progeny-arrays and segregation analyses (e.g. one allele was not detected in heterozygous individuals). However, this concerned fewer than 1% of the situations studied, and multilocus FIS estimates were hardly affected (results not shown). As already mentioned, null alleles are segregating in the populations studied, but this hardly affected the estimated selfing rates (see online electronic supplementary material). On the whole, estimates derived from population structure seem to only slightly over-estimate mean selfing rates.

Genetic diversity within populations and population size

The microsatellite loci used are moderately polymorphic, with gene diversity ranging from 0.2 to 0.6. However, these values are higher than those obtained at much more limited geographical scale using allozymes [START_REF] Dillon | The biogeography of sea islands: clues from the population genetics of the freshwater snail, Physa heterostropha[END_REF][START_REF] Jarne | The influence of self-fertilization and grouping on fitness attributes in the freshwater snail Physa acuta: population and individual inbreeding depression[END_REF]. The mean gene diversity (0.43) is not very different from that reported in Biomphalaria glabrata, the other outcrossing freshwater snail species studied using microsatellites (Mavárez et al. 2002a,b), but is higher than values reported in selfing species (see Table 4 in Mavárez et al. 2002a). This is expected because the effective population size decreases with the selfing rate (review in [START_REF] Städler | Population biology, genetic structure, and mating system parameters in freshwater snails[END_REF].

Estimates of genetic diversity were predicted to correlate with habitat openness and water regime (Table 2). Indeed, we observed more variation in rivers than in ponds. However, this might not be due to local population size because higher population densities were observed year-round in ponds. Note that this assumes that density correlates with local population size, which may not be true. A plausible explanation is that population size in rivers should be considered at the level of whole rivers. Similar estimates of genetic diversity were unexpectedly detected in permanent and temporary habitats (Web Table 5). One explanation suggested by the demographic survey is that large population size might be maintained in temporary habitats, despite drying out in summer. Of course the standing level of variation also depends on the past population size which is now discussed.

Comment citer ce document : Bousset, L., Henry, P.-Y., Sourrouille, P., Jarne, P (2004). Population biology of the invasive freshwater snail Physa acuta approached through genetic markers, ecological characterization and demography. Molecular Ecology, 13 (7), 2023-36. , DOI : 10.1111/j. 1365-294X.2004.02200.x This file is the post-print of: Bousset et al. Molecular Ecology (2004) 13:2023-2036doi: 10.1111/j.1365-294X.2004.02200.x 11 Population demography: bottlenecks Genetic signals of bottlenecks were detected in one, three and five populations under the SMM, TPM and IAM, respectively (Web Table 6). However, the tests were significant for the three models in two populations only (Lam1 and Mos1), both sampled around Montpellier. There is, therefore, limited evidence of bottlenecks: the populations studied probably did not experience a sudden decrease in size (with subsequent constant size) at the appropriate time scale for the tests performed (about 2Ne generations). However, it remains unclear how these tests are affected by fluctuating population size or migration, both likely in the situations studied. Fast increase after bottlenecks (see [START_REF] Appleton | Upstream migration by the invasive snail, Physa acuta, in Cape Town, South Africa[END_REF]; for data in P. acuta) may also dilute the bottleneck signal [START_REF] Nei | The bottleneck effect and genetic variability in populations[END_REF]. This might explain why no genetic signal of demographic variation was detected at global scale. At first glance, this might be considered surprising because introduced species are expected to exhibit limited variation due to bottlenecks at introduction. P. acuta was first described in Europe in 1805 (Garonne River, France), supposedly spread towards the Mediterranean region, and then slowly towards northern Europe [START_REF] Dillon | Populations of the European freshwater pulmonate Physa acuta are not reproductively isolated from American Physa heterostropha or Physa integra[END_REF]. Our data, therefore, suggest invasion by a large number of propagules, in agreement with some theories of biological invasions [START_REF] Williamson | Biological Invasions[END_REF], or successive invasions from the same genetic origin, presumably North America (L. Bousset & P. Jarne unpublished data).

The low number of significant genetic tests precluded a comparison with regard to habitat openness and water regime. However, comparisons were possible with demographic data: at the time scale considered, populations from ponds are no more likely to experience periods of low density than those from rivers. This contradicts genetic results on within-population polymorphism (based on the opposite demographic predictions). The sole extinction events were observed in a river (Lam1), where the flat, cemented riverbed did not offer refuges to snails during floods [START_REF] Henry | Dynamique des populations et évolution du système de reproduction. Etude chez le gastéropode Physa Acuta[END_REF]. Interestingly this is one of the two populations in which a strong genetic signal of bottlenecks was detected. The demographic survey also indicates that populations from temporary habitats did not experience marked bottlenecks. No extinction followed drying out in summer. The absence of demographic difference between temporary and permanent habitats (Web Table 5) was confirmed using a specifically designed capture-recapture monitoring of density in Lam12 during summer 2001 [START_REF] Henry | Dynamique des populations et évolution du système de reproduction. Etude chez le gastéropode Physa Acuta[END_REF]. The site dried out completely, and up to 99% of individuals died between June and October. Following the first autumn rains, the density was still high (17 individuals/m). This is probably a consequence of snail behaviour: they buried themselves in the muddy waterbed allowing significant survival during drought [START_REF] Henry | Dynamique des populations et évolution du système de reproduction. Etude chez le gastéropode Physa Acuta[END_REF]. Migration is unlikely to explain this result since the closest source of immigrants was 1.2 km upstream. In agreement with these results, the temporal genetic analysis indicated limited fluctuation in allelic frequencies between samples taken before and after summer drought. This further suggests that marked local environmental variation does not imply strong genetic fluctuation through sampling drift.

In conclusion, the environmental descriptors are poor predictor of bottlenecks, whether analysed through genetic or demographic variation. The main demographic difference, especially in relation to habitat openness, might be the occurrence of bottlenecks at population foundation (explaining the lower genetic variation) and low migration rates in closed habitats (see Discussion).

Migration

The range of pairwise FST estimates at the local scale (0.01-0.51) is consistent with values reported by [START_REF] Dillon | The biogeography of sea islands: clues from the population genetics of the freshwater snail, Physa heterostropha[END_REF] at similar geographical scale. At the local scale, isolation-by-distance was not detected when considering all populations. However, much more population differentiation was detected in ponds than in rivers (as expected), and estimates of Dσ 2 are about five times higher in rivers than in ponds (the regression slopes were significant different). An explanation might be that dispersal is more limited in ponds where it most likely derives from rare opportunities of transport by humans or mammals (sheep or wild boar Sus scrofa, bird vectors being unlikely here; Henry 2002). In rivers, downstream dispersal along the water current [START_REF] Clampitt | How fast is a snail pace? Active and passive dispersal of Physa integra in Douglas Lake, Michigan[END_REF] or active upstream migration [START_REF] Appleton | Upstream migration by the invasive snail, Physa acuta, in Cape Town, South Africa[END_REF][START_REF] Pointier | Introduction and spread of Thiara granifera (Lamarck, 1822) in Martinique, French West Indies[END_REF]) are probably more efficient ways of dispersing. A similar explanation might hold at the global scale at which more differentiation was also detected in ponds than in rivers. Birds might play a more significant role at this scale [START_REF] Santamaría | Waterbird-mediated dispersal of aquatic organisms: an introduction[END_REF].

What can be gained here from demographic data? From the monthly survey of snail size distributions (in rivers only), major events of immigration were detected in one, temporary population only (Lam1). Indeed Lam1 is best considered as a transient population with recurrent extinction-recolonization on a yearly basis, Comment citer ce document : Bousset, L., Henry, P.-Y., Sourrouille, P., Jarne, P (2004). Population biology of the invasive freshwater snail Physa acuta approached through genetic markers, ecological characterization and demography. Molecular Ecology, 13 (7), 2023-36. , DOI : 10.1111/j. 1365-294X.2004.02200.x This file is the post-print of: Bousset et al. Molecular Ecology (2004) 13:2023-2036doi: 10.1111/j.1365-294X.2004.02200.x 12 because the substrate offers no protection during floods. In other populations, immigration events could not be documented. Although the technique is limited to specific migration events (see Materials and methods), this is a first indication that migration proceeds through the exchange of a small number of migrants at a time in rivers. Complementary capture-mark-recapture analyses during flooding events [START_REF] Henry | Dynamique des populations et évolution du système de reproduction. Etude chez le gastéropode Physa Acuta[END_REF] are indicative of the same trend. In one experiment, 98% of marked individuals were displaced by flood but were recaptured at short distance (< 28 m), which presumably could be compensated for by active upstream dispersal. Even flooding in rivers does not necessarily cause massive displacement of populations, apart in peculiar habitats such as Lam1.

Conclusion

Combining approaches in population biology

Our study indicates that a pluralistic approach combining genetic and demographic data with environmental descriptors can be helpful when analysing the role of the four important factors considered here (see also, e.g. [START_REF] Whitlock | Some population genetic consequences of colony formation and extinction: genetic correlations within founding groups[END_REF][START_REF] Peacock | Dispersal in Pikas (Ochotona princeps): combining genetic and demographic approaches to reveal spatial and temporal patterns[END_REF]Charbonnel et al. 2002c;[START_REF] Chlyeh | Population genetics and dynamics at short spatial scale of Bulinus truncatus, the intermediate host of Schistosoma haematobium, in Morocco[END_REF]). The mating system can be inferred from both direct and indirect approaches. Mean selfing rates did not differ much, and it is likely that most inbreeding is due to selfing, especially at the local scale. However, the direct approach provides interindividual variation (Henry et al. submitted for publication) that might be of prime interest when studying the evolution of the selfing rate (see, e.g. [START_REF] Schultz | Individual variation in inbreeding depression: the roles of inbreeding history and mutation[END_REF]. Habitat openness influences the amount of genetic variation within populations, as already shown by Charbonnel et al. (2002a) in another freshwater snail, suggesting that even rough environmental characterization can be used to predict population variability. However, water regime can apparently not be used to predict this variation (but see Charbonnel et al. 2002a), perhaps because this trait does not capture essential features of variation in population size. Surprisingly, populations from permanent habitats do not pass through low densities less often than do those from temporary habitats at the time-scale studied. That limited evidence of genetic bottlenecks was gathered is compatible with the fact that even marked a reduction in water availability is not necessarily associated with demographic bottlenecks. A major conclusion with regard to the population biology of invasive species, such as P. acuta, is that those bottlenecks reducing genetic variation probably occur at population foundation, and that further demographic variation in population size might affect genetic variation to a limited extend. This is consistent with our most striking result, which is the lower genetic differentiation in rivers than in ponds. Bottlenecks (here at foundation) indeed increase genetic differentiation. A similar result was obtained in B. glabrata populations in Venezuela (Mavárez et al. 2002b), or in a marine snail when contrasting continuous and discontinuous habitats [START_REF] Johnson | Neighbourhood size and the importance of barriers to gene flow in an intertidal snail[END_REF], 1998). Similarly, [START_REF] Giles | Genetic differentiation in Silene dioica metapopulations: estimation of spatiotemporal effects in a successional plant species[END_REF] observed less differentiation among old than among young populations of a plant. The demographic data allowed us to refine the picture in rivers: flooding does not induce marked gene flow and massive events of migration were not detected which might explain why genetic differentiation is observed at short geographical scale. These data also revealed that some populations are transitory with recurrent recolonization, a pattern that cannot be detected through classical surveys of population genetic structure.

We are aware that the pluralistic approach followed here remains rather crude. More precise studies should conjointly estimate parameters. This has, for example, been done in a few studies for gene flow, as mentioned in the Introduction (see [START_REF] Rousset | Inferences from spatial population genetics[END_REF]. Alternatively, it would be interesting to have models of population genetic structure incorporating demographic parameters (e.g. population size, dispersal rate) in a more realistic way, or frameworks under which the likelihood of different models could be fitted to data and compared. Progress is made in this area (e.g. [START_REF] Estoup | Inferring population history from microsatellite and enzyme data in serially introduced cane toads, Bufo marinus[END_REF][START_REF] Leblois | Influence of mutationnal and sampling factors on the estimation of demographic parameters in a continuous population under isolation by distance[END_REF]. Our approach might also be improved if environmental variables were continuous, rather than binary (e.g. rivers vs. ponds). More sophisticated approaches of the relationship between genetic and environmental variables are also possible (see, e.g. [START_REF] Angers | Canonical correspondence analysis for estimating spatial and environmental effects on microsatellite gene diversity in brook charr (Salvelinus fontinalis)[END_REF]. Finally, the pluralistic approach has sometimes been perceived as problematic, because of the variety of time scales at which the different methods are working. For example, tests of bottlenecks on genetic data are concerned with events occurring a few Ne generations ago, whereas current estimates are derived from demography. The problem is not so much acute, as shown by results on dispersal [START_REF] Rousset | Inferences from spatial population genetics[END_REF]. A more optimistic perspective is that the pluralistic approach is an improvement over purely genetic or demographic approaches when analysing evolutionary forces acting on populations at various time scales. It remains though that a general framework reconciling population genetics and demography has still to be developed.

Comment citer ce document : Bousset, L., Henry, P.-Y., Sourrouille, P., Jarne, P (2004). Population biology of the invasive freshwater snail Physa acuta approached through genetic markers, ecological characterization and demography. Molecular Ecology, 13 (7), 2023-36. , DOI : 10.1111/j. 1365-294X.2004.02200.x This file is the post-print of: Bousset et al. Molecular Ecology (2004) 13:2023-2036doi: 10.1111/j.1365-294X.2004.02200.x 13 Comment citer ce document : Bousset, L., Henry, P.-Y., Sourrouille, P., Jarne, P (2004). Population biology of the invasive freshwater snail Physa acuta approached through genetic markers, ecological characterization and demography. Molecular Ecology, 13 (7), 2023-36. , DOI : 10.1111/j.1365-294X.2004.02200.x Comment citer ce document : Bousset, L., Henry, P.-Y., Sourrouille, P., Jarne, P (2004). Population biology of the invasive freshwater snail Physa acuta approached through genetic markers, ecological characterization and demography. Molecular Ecology, 13 (7), 2023-36. , DOI : 10.1111/j.1365-294X.2004.02200.x

Figure 2

 2 Figure 2 Differentiation between pairs of populations in rivers and ponds at local scale. Multilocus estimates of pairwise differentiation are plotted against the logarithm of the linear distance (D; in km). The corresponding RMA regression lines and the percentage of variation explained (r2) are indicated, with FST/(1 -FST) = 0.32 ln(D) + 0.55 and FST/(1 -FST) = 0.06 ln(D) + 0.07 in ponds and rivers, respectively.

Acknowledgements

We thank C. Chevillon, P. David, R.T. Dillon, F. Nijokou and J.-P. Pointier for snail sampling, C. Debain for help with the molecular analysis and J.-D. Lebreton for statistical advices. L. Bernatchez, N. Charbonnel, P.-A. Crochet and three anonymous referees provided insightful comments on the manuscript that significantly improved its quality. This project was supported by funds from INRA to L. Bousset, from CNRS and Bureau des Ressources Génétiques to P. Jarne, from the INVABIO program from the French Ministry for Ecology and Sustainable Development (MEDD) to P. Jarne and a fellowship form the French Ministry for Higher Education (MENRT) to P.-Y. Henry.

Supplementary material

The following material is available from http://www.blackwellpublishing.com/products/journals/suppmat/MEC/MEC2200/MEC2200sm.htm

Appendix A1

Table S1 Analysis of allelic segregation at ten microsatellite loci in 13 selfed families (seven offspring). Some (L2, L3, L8, L10, L12, L15, L18) were second laboratory generation offspring of individuals collected in March 2002 in a pond located on the CNRS campus in Montpellier. Other individuals were collected in March 2002 at the egg stage either in streams (Lam1a, Lam1b, Lez5), or in ponds (Vio8a, Vio8b, Vio12) located within a radius of 20 km around Montpellier (Table 1, Fig. 1 of main text). The parental genotype (allele sizes) is given per family and locus on the first line. The second line indicates allelic segregation among offspring (homozygotes for the smallest allele : homozygotes for the larg-est allele: heterozygotes). ***indicates significant deviation from Mendelian expectations.

Table S2

Allelic frequencies at eight microsatellite loci in the 24 populations of Physa acuta analysed. Values at Pac4 and Pac7 were obtained once null homozygotes were discarded. For locus AF108758, allele 407 indeed represents two bands of size 140 and 147 respectively (see 1 in the text above for details). Sampling dates are given in Table 1 of main text.

Table S3

Estimates of Wright's FST (below the diagonal) and associated P-values of homogeneity tests (above the diagonal; [START_REF] Goudet | Testing differentiation in diploid populations[END_REF] at six microsatellite loci among population pairs of Physa acuta (24 populations were studied). Sampling dates are given in Table 1 of main text. 96% of these tests were significant at the 0.05 level, and 93% remained significant after a sequential Bonferroni correction.

Table S4

Relationship between the mean (standard deviation) proportions of individuals excluded and reassigned per population and ecological characterization of habitats. See main text for details on habitat characterization. The 'Test' column indicates Mann-Whitney U test (n1; n2; U); P is the associated probability and = is followed by ' # ' when the predictions were fulfilled. Samples in the eight populations sampled twice (at t1 and t2) were pooled prior to analysis.

Table S5

Comparisons of the genetic and demographic parameters (defined in Table 3) between permanent and temporary habitats. Values are means with standard deviations into parentheses. See main text for details on the parameters estimated and habitat characterization. The tests were conducted at local scale only, since water regime was not determined at global scale. The 'test' column indicates either Mann-Whitney U test (n1; n2; U) or Fisher exact test F (n1; n2), and P is the associated probability. 'nt' holds for no test, either when testing was irrelevant (Mantel and Dσ 2 ) or not possible (no demographic data at global scale, not enough data for bottlenecks and extinctions). >, < and = are followed by ' # 'when the predictions of Table 2 of main text were fulfilled. Values in the 'bottlenecks' row indicate the number of significant tests under the infinite allele model (IAM), two-phase model (TPM) and stepwise mutation model (SMM), and the total number of tests performed. 'km' in the 'differentiation' row gives the mean geographic distance (standard deviation) between population pairs. Slope (a) values in the isolation by distance row are provided with their 95% confidence intervals generated by bootstrapping.

Comment citer ce document : Bousset, L., Henry, P.-Y., Sourrouille, P., Jarne, P (2004). Population biology of the invasive freshwater snail Physa acuta approached through genetic markers, ecological characterization and demography. Molecular Ecology, 13 (7), 2023-36. , DOI : 10.1111/j. 1365-294X.2004.02200.x This file is the post-print of: Bousset et al. Molecular Ecology (2004) 13:2023-2036doi: 10.1111/j.1365-294X.2004.02200.x 14 Table S6 Tests of mutation-drift equilibrium under the infinite allele model (IAM), the two-phase model (TPM) and the stepwise mutation model (SMM). The table gives the probability associated with Wilcoxon signed ranks test of the null hypothesis of equilibrium. 'Excess' and 'Deficit' (when P ≤ 0.05) indicate respectively a bottleneck and an expansion.

Supporting Information

Lydia Bousset, currently a researcher at the Institut National de la Recherche Agronomique in Rennes (France), worked as a postdoctoral fellow on snail population genetic structure and invasion biology. Pierre-Yves Henry's PhD focused on the evolution of mating systems in snails, and is currently studying invasive populations of birds (Princeton, USA). Patricia Sourrouille is laboratory engineer with expertise in DNA analysis. Philippe Jarne works on the evolution of subdivided populations, largely using DNA markers, and of mating systems.


David Ramirez

Mustafa A Kocak

Elza Erkip

Dennis E Shasha

Confidence Based Refusal (CBR) SafePredict SafePredict + CBR Amnesic SafePredict

Wolf

Beard Man …

Observation

Crowd of experts (i.e., algorithms) are asked to predict. Dummy expert always refuses to predict.

Problem Setup

Introduction References

Machine learning and prediction algorithms are the building blocks of automation and forecasting.

Algorithms benefit from a lower error rate.

Definitions

Algorithm Properties

Def. A meta-algorithm is valid if, as 𝑇 * → ∞, average expected loss ≤ target error rate. Def. A meta-algorithm is efficient if, as 𝑇 * → ∞, refusals occur only a finite number of times.

SafePredict, a meta-algorithm, takes predictions from underlying algorithms and decides whether or not to predict with them.

Prediction ො 𝑦 𝑃,𝑡 or refusal ො 𝑦 𝐷 suffer a loss 𝑙 𝑃,𝑡 , 𝑙 𝐷 ∈ [0,1].

Mistakes are costly, but we learn by observing.

Safe-Predict is valid and efficient!

Guaranteed with no assumptions on data or underlying experts, but asymptotic in the number of non-refused predictions.

Experimental Results

Randomly permute data, choose first 10k points for experiment. Target error rate 𝜖 = 0.05.

Random label permutation introduced every 2k points (i.e., artificial change points).

Main Results

  𝑡 = time index, 𝑇 = total observations, 𝜂 = learning rate 𝑇 * = σ 𝑡=1 𝑇 𝑤 𝑃,𝑡 , expected predictions 𝐿 𝑇 * = σ 𝑡=1 𝑇 𝑙 𝑃,𝑡 𝑤 𝑃,𝑡 , expected cumulative loss 𝑉 * = σ 𝑡=1 𝑇 𝑤 𝑃,𝑡 𝑤 𝐷,𝑡 , variance for number of predictions 𝑤 𝑃,𝑡+1 = 𝑤 𝑃,𝑡 𝑒 -𝜂𝑙 𝑝,𝑡 𝑤 𝑃,𝑡 𝑒 -𝜂𝑙 𝑝,𝑡 +𝑤 𝐷 𝑒 -𝜂𝜖 weight shift rule

  SafePredict is guaranteed valid for any 𝑃. Particularly

	𝐿 𝑇 * 𝑇 * -𝜖 = 𝑂	𝑉 * 𝑇 * =	𝑇 * . 1	𝑡→∞ Theorem 2.-If lim sup	𝐿 𝑃,𝑡

Theorem 1.-With learning rate 𝜂 = Θ( 1 𝑉 * ), 𝑡 < 𝜖 and 𝜂𝑇 → ∞, then SafePredict is efficient.

Online prediction setup with refusal option.


Rabih Haddad

Eunika Mercier-Laurent

Integrated System for students' Evaluation Using KM Approach

Keywords: Higher Education, International Students, Knowledge Management, Text Mining, and Speech Recognition

With the number of international students increasing globally and the mobility of students is becoming a condition to secure a good job and to gain a shining career, evaluating candidates prerequisites is becoming challenging. This paper presents how knowledge management approach using AI techniques could help academic institutions in the evaluation of international students profiles by providing an adapted methodology. This methodology implemented in the proposed system will help institutions gain more time in processing students files, provide accurate evaluation of candidates by taking their cultural background into consideration and avoid human errors.

Introduction

France hosts each year international students willing to pursue their higher education in different fields. Their presence compensates for the relative lack of talented French students. The state and the French educational organizations want to increase the number of students to improve the position of the French educational system vis-a-vis the Anglo-Saxon system, among others.

More than 300,000 students annually from 180 different countries got enrollment in the French Universities and Graduate Schools. These academic institutions receive thousands of applications of the students who have completed their high school, bachelor, or masters degrees. This diversity of students profiles and their different academic background make accurate assessment an extremely challenging task. Up to now, the prior learning assessment of candidates and the processing of their applications are done manually and hence these tasks are time consuming and involve a lot of human potential errors in the selection. In addition, the distinctiveness of the French education system especially with its "Grandes Ecoles" that are unique, complicates the task of evaluation. International standard exams like SAT, GMAT, GRE are insufficient to conduct a full assessment.

What is missing? As per our knowledge existing systems lack of considering criteria such as cultural aspects, level of education in each country, motivation, real meaning of motivation letter, and emotional intelligence. In the light of the current state of the art and on our knowledge, there is no automated intelligent system that performs a multi-criterion evaluation of international students by multi-media and multi-modal interaction with them. Knowing the proposed programs, computer trends and needs of students, this system should also propose an adjustment of programs, according to the students level. Here comes the need for automated multi-criteria system for the evaluation of students knowledge and motivation.

Many researchers and universities have worked on several systems to evaluate the students profiles to provide coherent admission results and to process the maximum number of applications possible. One of these approaches is to compare the profiles of new applicants with those who have similar profiles and have already validated their academic programs. A second approach was based on ranking of applicants by using the available historical data and predictive analysis to detect the risk of admitting those candidates. Athors use these data to orient stduents toward a specifcied major or domain. In my opinion, all of these approaches do not serve to solve the main problem presented in this paper as they do not provide a subjective and adapted assessment exercises. Also, those systems do not evaluate international students attended universities and their learning outcomes. In addition, the decision support system implemented do not use the latest technology.

In this paper, we are going to present the related work, research methodology, challenge, and the proposed system for evaluating the stduents' profile using knowledge management.

Challenge

To remove this lock and define an effective system architecture, a deep understanding of the admission principles and related contexts is mandatory. Existing experience and explanation of the admission problem from the educative point of view must also be considered.

Today applying to any degree seeking program in France or anywhere in the world nationally or abroad requires that candidates should undergo an admission procedure to assess the profile of candidates and to announce the final decision (admission/refusal of candidates). Admission systems vary from one country to another and from one institution to another. The characteristics of each country and institution shape the admission system, however there is a big percentage of communality between these systems as they require the same traditional documentation, evaluation, information: admission and languages proficiency exams, interviews, CVs, transcripts, motivation letters, and recommendations letters.

Admission Systems

Up to now, all institutions need the candidate information and exams results to decide of the admission status of candidates. The admission procedure that is widely used worldwide and in France is described as follows:

-Students apply online on the institution website by supplying all the relevant and requested documents.

-Admission teams process the files following the order:

• Relevance of the candidate to the requested major

Knowledge Blocks

As an entry point to understanding the admission system, we should look at 2 major knowledge blocks that contribute to the relevant evaluation of students profiles: the Curriculum Vitae (CV) and the online interview. The CV still is an important document that helps the admission committee identify important information about the candidates and provide knowledge on students academic and career path. The online interview helps the admission committee to detect motivation and validate the coherence and genuineness of the candidates vis-a-vis their CVs and profiles. For this purpose, the online interviews will be registered and rerun for the offline evaluation.

Curriculum Vitae (CV)

This block is very essential in the evaluation process. In the current systems the CV students are not taken into consideration as the evaluation logic only depends on the profiles of previous students who have succeeded a certain curriculum. Hence it depends on a comparative mechanism that might be valid for students coming from the same background but might fail for a diversified group of students.This block should provide the below information about each candidate:

-Basic: to detect the country of origin of each candidate, age and gender. The country will be a crucial factor in the adapted evaluation since a cultural impact matters here.

-Academic background: to detect the institution attended by the candidate and the highest degree obtained and the number of academic years after high school. This will help also to compile a list of academic institutions worldwide that will be ranked based on students success after enrollment and pursuing of classes.

-Professional experience: to verify the experience and skills acquired during this experience and its relevancy to the degree obtained. This will help also to compile a list of companies worldwide that will be ranked based on students success after enrollment and pursuing of classes.

Beside the direct knowledge that will be extracted from CVs, text mining procedures will be applied to derive knowledge from the unstructured text by merging all the above listed information. We will apply statistics, analytics, semantic and natural language processing algorithm and output of this exercise could be a weighted mark that aggregate CV main parts.

Online Interview

This exercise will serve in providing several types of knowledge on candidates.

The online interview will be used to evaluate the English language level of candidates, their motivation, their capability to present themselves and present a coherent project of life, and some easy behavior aspects. The following 4 groups of knowledge could be extracted and evaluated from pre-registered video interviews:

-Detect the candidates oral ability by evaluating the first couple of minutes of their interview.

-Analyze the candidates behavior in the video in terms of self-confidence and coherence.

-Evaluate the candidates answers to the interview questions to detect their motivation and relevance.

The idea is to retrieve the audio files from the registered interviews. The following techniques could be used to have a precise evaluation: speech recognition, Large Vocabulary Continuous Speech Recognizers, Speech analytics and Semantic Interpretation for Speech Recognition. The output of this simulator could be a weighted mark on a scale of 5 (poor, fair, good, very good, excellent for example). This mark could be an aggregate of the language, motivation, behavior, and character. There is a possibility also to convert to text to apply the text mining techniques.

Proposed System

The comprehension of the nature and contexts of the elements leading to the correct evaluation will guide the choice of knowledge models and processing methods. The proposed architecture will contain several communicating building blocks. Working on the algorithm requires treating a list of modules that build up the main architecture. -Exploration of the elements gathered during collection phase and comprehension of the relations in systems components. Collected elements include information found during the educational research part. Exploration should include knowledge discovery and required analysis, as well as behavioral detection. The architecture will be adapted based on the educational research results.

-Generation of an adequate exam. The exam should consider the results found in the previous research and the academic profile of each candidate.

-Generation of the GAAF (Global Admission Acceptance Factor). This is the specific measure of student knowledge and capacity, that may vary according to the requirements.

Conclusion

In this paper, the integrated system of students evaluation using knowledge management approach is presented. The main aim is to assess how KM can help in a such system. By presenting and analyzing the 2 major knowledge blocks that constitute the evaluation system, we will be able to obtain an architecture that leads to an adapted evaluation of candidates. Also, this analysis will help us to validate the proposed solution using real cases and integrated feedback experience. The advantage of the proposed exam is that it does not assess only the aptitude and the knowledge of the candidate in a certain domain. It goes beyond the instantaneous evaluation of students to assess the experience, the skills acquired and the behavior in a multinational environment. The next step will be building up 2 simulators based on the CV text mining block and the interview evaluation block. These 2 simulators will be tested on hundreds of students applications we own to verify their outputs and validate the proposed algorithm. These 2 simulators will be crucial to building up the architecture of the proposed system and test in real case scenarios.

Fig. 1 .

 1 Fig. 1. Current Admission Cycle

Fig. 2 .

 2 Fig. 2. System Principles

  Exams conduction to detect knowledge and practice • Financial status (mainly Anglo-Saxon institutions)

	-Results announcements

• Candidates high school or bachelor grades • Candidates experience and skills • Interview conduction (remote or face to face) to detect: genuineness, motivation, and capacity •

5

  References 1. Simon Fong & Robert P. Biuk-Aghai An Automated University Admission Recommender System for Secondary School Students ICITA 2009 2. Heba. M. Rashad & Walid Mohamed Aly & Osama Fathy Hegazy. An Educational Data Mining System for Advising Higher Education Students World Academy of Science, Engineering and Technology 2013 3. Khalid A. Fakeeh Decision Support Systems (DSS) in Higher Education System International Journal of Applied Information Systems (IJAIS) 2015 4. Rajan Vohra & Nripendra Narayan Das Intelligent Decision Support Systems For Admission Management In Higher Education Institutes IJAIS 2011