Aliou Dia
email: adia@sb-roscoff.fr

L Guillou

S Mauger

E Bigeard

D Marie

M Valero

Christophe Destombe
email: destombe@sb-roscoff.fr

SPATIOTEMPORAL CHANGES IN THE GENETIC DIVERSITY OF HARMFUL ALGAL BLOOMS CAUSED BY THE TOXIC DINOFLAGELLATE ALEXANDRIUM MINUTUM

Keywords: population genetics, bloom dynamics, resting cyst, linkage disequilibrium, sexual reproduction, clonality minutum spatiotemporal genetic diversity Dia_et al_suppinfo.doc; contains datasets

Organisms with sexual and asexual reproductive systems benefit from both types of reproduction. Sexual recombination generates new combinations of alleles whereas clonality favors the spread of the fittest genotype through the entire population. Therefore, the rate of sexual versus clonal reproduction has a major influence on the demography and genetic structure of natural populations. We addressed the effect of reproductive system on populations of the dinoflagellate A. minutum. More specifically, we monitored the spatiotemporal genetic diversity during and between bloom events in two estuaries separated by 150 km for two consecutive years. An analysis of population genetic patterns using microsatellite markers revealed surprisingly high genotypic and genetic diversity. Moreover, there was significant spatial and temporal genetic differentiation during and between bloom events. Our results demonstrate that (1) interannual genetic differentiation can be very high,

(2) estuaries are partially isolated during bloom events, and (3) genetic diversity can change rapidly during a bloom event. This rapid genetic change may reflect selective effects that are nevertheless not strong enough to reduce allelic diversity. Thus, sexual reproduction and/or migration may regularly erase any genetic structure produced within estuaries during a bloom event.

Introduction

The demography and genetics of natural populations can vary according to the rate of sexual versus clonal reproduction. These two modes of reproduction occur simultaneously in many eukaryotes. These species can benefit both from the advantage of sexual reproduction, which lies in the genetic recombination that it generates, and from the theoretical short-term demographic advantage of clonal reproduction [START_REF] Smith | Polymorphism in a varied environment: how robust are the models[END_REF]. The relative contribution of sexual and asexual reproduction can vary within a species depending on environmental conditions [START_REF] Eckert | The loss of sex in clonal plants[END_REF][START_REF] Silvertown | The evolutionary maintenance of sexual reproduction: evidence from the ecological distribution of asexual reproduction in clonal plants[END_REF]. For example in aphids, sexual reproduction is associated with the production of resting eggs that can survive harsh conditions, whereas populations living in milder environments may reproduce asexually throughout the whole year [START_REF] Rispe | Models of sexual and asexual coexistence in aphids based on constraints[END_REF]. Sexual reproduction thus seems required to produce a resistant form, regardless of the genetic variability generated. In the freshwater microcrustacean Daphnia pulicaria that practices facultative sexual reproduction (alternation between parthenogenesis and sexual reproduction) during the growing season, selection on clonal lines results in an increase in genotypic adaptation and an erosion in clonal diversity [START_REF] Deng | Change of genetic architecture in response to sex[END_REF]. The over-representation of certain clonal genotypes at the end of the growing season leads to greater genetic structuring and linkage disequilibrium. Similarly, most plankton species alternate between an active growing phase, during which the number of cells increases rapidly, and another phase, generally involving sexual reproduction, during which cells are in a dormant state. During the asexual reproduction phase, fitness differences among clonal lineages may cause rapid changes in genotype frequencies. In the cyclically parthenogenetic rotifer species Brachionus plicatilis, there is evidence that clonal selection occurs during the parthenogenetic phase [START_REF] Gómez | Sex, parthenogenesis and genetic structure of rotifers: microsatellite analysis of contemporary and resting egg bank populations[END_REF]. However, sexual reproduction as well as dormant stages may slow down the response to directional selection and may act to maintain genetic variation when selection fluctuates [START_REF] Hairston | Phenotypic variation in a zooplankton egg bank[END_REF].

Thus, in the freshwater copepod Diaptomus sanguineus, the dormant cells may act as a reservoir of genotypes (similar to a seed bank) produced in past environments, but they also may interact as a part of the selection-response dynamics in the current environment [START_REF] Hairston | Phenotypic variation in a zooplankton egg bank[END_REF]).

An extreme case of alternation of an asexual growing phase and a dormant sexual phase is exemplified by unicellular phytoplankton bloom species involved in harmful algal blooms (HABs). Compared to multicellular organisms, these unicellular organisms are characterized by a ubiquitous distribution, suggesting very large population sizes and broad dispersal abilities [START_REF] Cloern | Tidal stirring and phytoplankton bloom dynamics in an estuary[END_REF][START_REF] Finlay | Cosmopolitan metapopulations of free-living microbial eukaryotes[END_REF][START_REF] Norris | Pelagic species diversity, biogeography, and evolution[END_REF]. Nevertheless, dispersal of marine phytoplankton is a hotly debated issue and published results are subject to controversies. Studies on marine phytoplankton dispersal differ vastly with respect to study scale, sampling strategy (e.g. sampling few individuals per global geographic region; population samples) and the resolution of the characters or markers used (e.g. morphological markers; highly variable, microsatellite-type molecular markers). For example, using morphological criteria, [START_REF] Cermeno | Controls on diatom biogeography in the ocean[END_REF] argue that the biogeographical distribution of fossil diatom genera is ubiquitous and that these taxa thus have no geographic structure. However, more recent studies using microsatellite markers demonstrate that there is genetic differentiation within a single species, the toxic diatom Pseudo-nitzschia pungens, on a very large scale (i.e. samples from the Atlantic and Pacific Oceans) [START_REF] Casteleyn | Limits to gene flow in a cosmopolitan marine planktonic diatom[END_REF].

The issue of connectivity between populations remains unresolved because there is little information on the distance that a single phytoplankton cell can cover and because there are only a few studies on how the marine physical environment influences dispersal. Exploring these factors is necessary for a better understanding of the ecology and evolution of plankton organisms [START_REF] Palumbi | Reproductive isolation, genetic divergence, and speciation in the sea[END_REF]. Over the past several years, more and more studies have focused on spatiotemporal differentiation in dinoflagellate populations. For example, in the Gulf of Maine, genetic differentiation was found between two subpopulations investigated during offshore blooms, corresponding to the beginning and the end of a bloom event (which lasts about 2 months), but in different geographic locations (northern and southern, separated by hundreds of km) [START_REF] Erdner | Diversity and dynamics of a widespread bloom of the toxic dinoflagellate Alexandrium fundyense[END_REF]. Other work [START_REF] Richlen | Extensive genetic diversity and rapid population differentiation during blooms of Alexandrium fundyense (Dinophyceae) in an isolated salt pond on Cape Cod, MA, USA[END_REF] shows spatiotemporal differentiation in the estuaries of Massachusetts (Nauset Marsh system and the Gulf of Maine) at two sites, but only one site was sampled throughout the whole bloom event (from beginning to end), precluding any comparison between dates of the same bloom at different sites. Whatever their scale, these previous studies demonstrate genetic differentiation between sites and high genetic diversity within a given bloom event. This differentiation of bloom populations and their high genetic diversity leads to questions on the role of the two life-cycle phases on population dynamics and the importance of sexual reproduction relative to clonal reproduction.

The dinoflagellate Alexandrium minutum [START_REF] Halim | Alexandrium minutum nov. g., nov. sp., a dinoflagellate causing red water[END_REF]) is one of the main species involved in paralytic shellfish poisoning (PSP) blooms off the coasts of France. This microalga produces saxitoxins that can accumulate along the trophic food web. They produce PSP events that cause cases of human poisoning, typically by ingestion of contaminated shellfish. This species shows an alternation of asexual and sexual phases. The vegetative cell is haploid and sexual reproduction takes place when the environmental conditions become unfavorable [START_REF] Figueroa | Interactive effects of salinity and temperature on planozygote and cyst formation of Alexandrium minutum (Dinophyceae) in culture[END_REF]. Sexual reproduction is the result of the fusion between two gametes, forming a planozygote [START_REF] Probert | Morphological details of the life history of Alexandrium minutum (Dinophyceae)[END_REF]. This stage changes into a resting cyst that sinks to the sediment (like seeds in a seed bank) [START_REF] Wyatt | Notes on Alexandrium population dynamics[END_REF]. After a dormancy period of several months [START_REF] Garcés | Relationship between vegetative cells and cyst production during Alexandrium minutum bloom in Arenys de Mar harbour (NW Mediterranean)[END_REF][START_REF] Figueroa | Comparative study of the life cycles of Alexandrium tamutum and Alexandrium minutum (Gonyaulacales, Dinophyceae) in culture[END_REF], cysts may germinate and give rise to vegetative haploid cells that reproduce asexually in the water column.

The objective of this study was to study the effect of asexual and sexual reproduction on the population genetic structure of this toxic dinoflagellate A. minutum on a small scale, both in space and in time. We used microsatellite markers to analyze monoclonal cultures of A. minutum isolated from samples taken at two sites located along the French coast of the English Channel, separated by roughly 150 km, for two consecutive annual bloom events and at three different periods during each bloom event. We specifically addressed the following questions: (1) To what degree are different estuaries within the same geographic region connected? (2) What role does sexual reproduction play in the dynamics of the bloom populations? (3) Finally, is there any temporal genetic structuring during bloom events?

Materials and Methods

Sampling sites and strain collection

More than 1240 strains of dinoflagellates (different species) were collected in the Penzé estuary (48°37'37.57"N, 3°57'13.17"W) and the Rance estuary (48°31'49.61"N, 1°58'21.81"W, Table 1). These two estuaries are separated by approximately 150 km (Fig. 1).

A. minutum blooms occurred concomitantly in both ecosystems in early summer. In 2010, blooms extend from 17 May to 12 July in the Rance estuary (maximum density of 4.2 x 10 4 cells/L -1 observed on June 22) and from 2 June to 7 July in the Penzé estuary (maximum density of 3.7 10 5 cells/L -1 observed on 8 June). In 2011, blooms lasted from 11 May to 20 June in Rance (maximum density of 4.3 10 5 cells/L -1 observed on 27 May) and from 1 June to Plankton samples were collected from these two sites during the developmental phase of the bloom (beginning), during the maintenance phase (middle) and during the decline phase (end of the bloom event) whenever possible in each of two study years (2010 and 2011). All strains were obtained by micropipetting one single cell into fresh medium; 530 of these strains survived and 265 strains of A. minutum were genotyped. They were maintained at 19°C with 70 µE.m 2 .s -1 of light in a 12:12 light:dark cycle. The medium F/2 (Marine Water Enrichment Solution, Sigma) was prepared using autoclaved natural seawater from the Penzé estuary. It was collected at least 3 months prior to use and stored in the dark. Strains used in this study (see supplementary Table S1) have been deposited at the Roscoff Culture Collection (www.sb-roscoff.fr/Phyto/RCC).

Flow cytometry

The DNA content of each monoclonal strain was checked by flow cytometry on whole A. minutum cells fixed in ethanol. A 250 µL aliquot of each culture was centrifuged at 3000 rpm for 10 min. The supernatant was removed, cells were then re-suspended by vortexing in 250 µL of 70% ethanol and kept overnight at 4°C. Cells were collected by centrifugation at 3000 rpm for 10 min to remove ethanol and then resuspended in 250 µL of PBS. Triton X-100 (0.1% final), RNase A (0.01%) and Propidium Iodide (PI, 30 µg.mL -1 ) were added.

Samples were incubated at 37°C for 30 min before flow cytometry analysis, performed using a FACS Canto II (Becton Dickinson, San Jose, CA, USA) flow cytometer equipped with a 488 nm laser and the standard filter setup. The DNA content of A. minutum strains was evaluated by comparison with Human Blood Cells (HBCs, 2C = 6.4 Mbp). Isolated nuclei of human blood cells were obtained by resuspending 10 µL of blood in 1 mL of NIB buffer [START_REF] Marie | DNA, RNA analysis of phytoplankton by flow cytometry[END_REF]. Then, 10 µL of this suspension was added to 250 µL of A. minutum culture re-suspended in PBS buffer before staining. Then, all cultures were analyzed by flow cytometry without HBC and mean florescence of PI were compared to check for ploidy level differences.

DNA extraction, strain identification and genotyping

For DNA extraction, strains were collected during their exponential growth stage (strains listed Table 1). DNA was extracted using either the CTAB method [START_REF] Lebret | Genetic diversity within and genetic differentiation between blooms of a microalgal species[END_REF] or the Nucleospin 96 Plant kit (Macherey-Nagel, Düren, Germany), according to the manufacturer's instructions, using 5-10 mg of dried tissue resuspended in 100 µL elution buffer.

Strains were screened using the intergenic region of ribosomal DNA (ITS1, 5.8S and ITS2). PCR mixes (14 µL in total) included 20 ng of DNA, 1X GoTaq Flexi green buffer (Promega, Madison, WI, USA), 150 µM of each dNTP (Thermo Fisher Scientific Inc., Waltham, MA, USA), 20 pmol of the forward primer 329F (5′-GTG AAC CTG CRG AAG GAT CA-3′, complementary reverse of the eukaryote reverse primer 329-R (Moon-van der [START_REF] Der Staay | Oceanic 18S rDNA sequences from picoplankton reveal unsuspected eukaryotic diversity[END_REF], 20 pmol of the reverse primer DIR-R (5′-TAT GCT TAA AAT TCA GCA GGT-3′ [START_REF] Scholin | Identification of group-and strain-specific genetic markers for globally distributed Alexandrium (Dinophyceae). I. Rflp analysis of SSU rRNA genes[END_REF], and 1.25 U GoTaq® Polymerase (Promega).

Amplification was carried out in a DNA Engine Peltier Thermal Cycler (Bio-Rad, Hercules, CA, USA) with the following cycling conditions: an initial denaturation at 95°C for 5 min, followed by 35 PCR cycles (denaturation at 95°C for 45 s, annealing at 53°C for 45 s, and extension at 72°C for 45 s), followed by a final extension at 72°C for 10 min. The PCR products were purified using the BigDye ® Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA) for subsequent bidirectional sequencing using a capillary sequencer type ABI 3130XL (Applied Biosystems).

The strains were genotyped using the 12 microsatellite markers developed by [START_REF] Nagai | Development of microsatellite markers in the toxic dinoflagellate Alexandrium minutum (Dinophyceae)[END_REF]. Amplification of microsatellite loci was carried out in a 10 µL final volume with the following components: 20 ng DNA, 1x GoTaq® Flexi buffer (Promega), 2 mM MgCl 2 , 150 µM each dNTP (Thermo Fisher Scientific Inc.), 0.8 µg.µL -1 bovine serum albumin, 30 pmol fluorescent-labeled forward primer (FAM, VIC, NED or PET), 30 pmol reverse primer, and 0.35 U GoTaq® Polymerase (Promega). PCR amplifications were carried out in a Bio-Rad DNA Engine Peltier Thermal Cycler using the following cycling conditions: an initial denaturation at 95°C for 5 min, followed by 10 PCR cycles (denaturation at 95°C for 30 s, annealing at 60°C for 30 s and extension at 72°C for 30 s) followed by 28 cycles (denaturation at 95°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 30 s), followed by a final extension at 72°C for 10 min. Then, 2 µL of PCR products, diluted 1:10, was added to 10 µL of loading buffer containing 0.5 µL of SM594 size standard [START_REF] Mauger | A simple and cost-effective method to synthesize an internal size standard amenable to use with a 5-dye system[END_REF]) and 9.5 µL of Hi-Di formamide (Applied Biosystems). The loading mix was denatured at 95°C for 3 min and run in an ABI 3130 XL capillary sequencer (Applied Biosystems) equipped with 50 cm capillaries. Genotypes were scored manually using GeneMapper version 4.0 (Applied Biosystems).

Genetic analyses

To increase sample size, close sampling dates were pooled (i.e. PZ10D, PZ11B and RC11D, Table 1) and ultimately only 11 samples with a minimum sample size of 15 strains were used in the genetic analysis (i.e. RC10D was excluded from the analyses; see Table 1).

Genetic diversity and multilocus genotype diversity

Because strains of A. minutum are haploid, the frequencies of null alleles were estimated by direct observation. After two re-amplifications, strains for which more than half of the loci did not amplify correctly were removed from the dataset. The absence of amplification at loci in other strains was considered as a null allele. Standard measures of genetic diversity (mean number of alleles per locus (Na), Nei's unbiased estimator of genetic diversity (H s ; Nei 1987), and allelic richness (Ra)) for each sampling date and site were calculated using Fstat (version 2.9.3.2; Goudet 2001). Allelic richness was estimated after randomly subsampling each sample (10,000 randomizations) to standardize the minimum sample size among populations.

Test for differences among groups, Fstat [START_REF] Goudet | Fstat, a program to estimate and test gene diversities and fixation indices (version 2.9[END_REF]) was used to test the difference between the H s values obtained at the beginning phases (PZ11A, RC10A, RC11A) and at the end phases of bloom events (PZ10D, PZ11D, RC11D). Genclone (Arnaud-Haond et al. 2005) was used to test the efficiency of the microsatellite marker set to discriminate the maximum clonal diversity available in the data set and to calculate the number of distinct multilocus genotypes observed (G).

Linkage Disequilibrium

Linkage disequilibrium (LD) was assessed using a single multilocus measurement of LD that is provided by the association index r̅ d [START_REF] Brown | Multilocus structure of natural populations of Hordeum spontaneum[END_REF]modified by Agapow & Burt 2001) and was computed using MultiLocus ver 1.2. [START_REF] Agapow | Indices of multilocus linkage disequilibrium[END_REF]. Significance tests were based on comparisons of the observed value to those of randomized datasets generated from 1000 permutations [START_REF] Burt | Molecular markers reveal cryptic sex in the human pathogen Coccidioides immitis[END_REF]. The global P-value was calculated using a generalized binomial procedure to combine independent data [START_REF] Teriokhin | On the power of some binomial modifications of the Bonferroni multiple test[END_REF][START_REF] De Meeûs | MultiTest V.1.2, a program to binomially combine independent tests and performance comparison with other related methods on proportional data[END_REF].

Genetic differentiation

F st values were calculated between all pairs of populations using GENETIX 4.05 (Belkhir et al., 1996(Belkhir et al., -2004)), their significance was assessed using 1.000 permutations and D (D est ;[START_REF] Jost | G(ST) and its relatives do not measure differentiation[END_REF] was estimated with SMOGD 1.25 [START_REF] Crawford | SMOGD: software for the measurement of genetic diversity[END_REF]. Different levels of comparison were thus considered: among dates during a bloom event, among dates within a site, and between sites. These data were summarized and statistically tested with a principal component analysis (PCA) performed using PCA-GEN software (version 1.2; Goudet 1999).

The statistical significance associated with each axis was evaluated after 10,000 randomizations.

The partitioning of genetic variation at different hierarchical levels was examined using molecular analyses of variance (AMOVA) in Arlequin 3.5.1.2 [START_REF] Excoffier | Arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under Linux and Windows[END_REF].

There was only one sampling date during the 2010 bloom in Rance (Table 1); it was therefore only possible to conduct the two following analyses. First, variation was assessed on the 2011 samples by determining the variance component attributable to between-sites differentiation and to among in-bloom phases within a site. Second, variation was examined in more detail only at Penzé by determining the variance component attributable to between-years differentiation and to among in-bloom phases within years. The significance (P < 0.05) of the fixation indices was evaluated based on 10,000 permutations.

Results

Isolation and identification of strains

Of the 1240 isolated cells, we obtained 530 usable strains for this study (i.e. 43% success rate). The survival rate by sampling date ranged from 27% to 66%. Of these 530 monoclonal strains, 364 (66%) were assigned to A. minutum showing a unique haplotype corresponding to 100% similarity with a 550 bp of the rDNA sequence (covering ITS1, the 5.8S rRNA gene, and ITS2) [START_REF] Mccauley | Biogeographic analysis of the globally distributed harmful algal bloom species Alexandrium minutum (Dinophyceae) based on rRNA gene sequences and microsatellite markers[END_REF][START_REF] Casabianca | Population genetic structure and connectivity of the harmful dinoflagellate Alexandrium minutum in the Mediterranean Sea[END_REF] were selected (Table 1). Indeed, this unique sequence corresponds to an A. minutum sequence (AM1 strain) previously published in GenBank (EU707466). The comparison of A. minutum DNA content shows that whole cells of A. minutum analyzed contain the same level of ploidy (32 Mbp, Fig. S1).

Genetic diversity

After re-amplifying strains with dubious amplification, 5 of the 12 loci (Aminu39, Aminu44, Aminu10, Aminu15 and Aminu29) developed by [START_REF] Nagai | Development of microsatellite markers in the toxic dinoflagellate Alexandrium minutum (Dinophyceae)[END_REF] were removed from the dataset. We also removed strains for which more than half of the loci did not amplify correctly. The final dataset included seven loci amplified on 265 individual strains (Table S2).

For these loci, the frequency of null alleles varied from 8% for locus Aminu11 to 22% for locus Aminu20. The efficiency of the microsatellite loci to discriminate the maximum clonal diversity available in the sample is given Fig. S2. Of the 265 strains analyzed, 265 (100%) had unique seven-locus genotypes; there were therefore no repeated genotypes in any of the analyzed strains.

The PCR products of these seven microsatellite markers showed one single band, i.e. one allele. All loci were polymorphic. The genetic diversity indices are given in Table 2. The number of alleles varied among bloom populations, with some populations having twice as many alleles as other populations (cf. RC11A and RC10A, Table 2), and with sampling effort.

Allele richness, corrected for sampling size, varied from 6.49 (RC11A) to 9.01 (RC11C). The per-locus and per-population numbers of alleles (N a ) ranged from 3 (locus A41) to 17 (locus A20) alleles. Whatever the sampling date during bloom events, Penzé and Rance showed high genetic diversities, with H s values ranging from 0.79 to 0.88. These genetic diversity values did not differ between the beginning and the end phases of a bloom event within an estuary (permutation test of comparison among groups; P-value=0.647).

Tests of linkage disequilibrium revealed an overall P-value of 0.525. Of the 11 spatiotemporal samples, only one showed significant r̅ d (i.e. linkage disequilibrium estimator) values. This sample from Penzé (PZ11D) had a P-value of 0.047, with r̅ d values of 0.068 (Table 3).

Genetic differentiation

The results of the PCA (Fig. 2) show a significant overall F st value of 0.073. The first two PCA axes were significant and explained 44% of the variance: axis 1 accounts for 26.39% of the variance (F st = 0.019, P=0.001) and axis 2 accounts for 17.65% of the variance (F st =0.013, P=0.011). Axis 1 separates samples from the beginning of the 2010 bloom (PZ10A-RC10A) from the rest of the samples (Fig. 2). Axis 2 separates samples according to the sampling date during a bloom and their geographic origin. Samples from the beginning of the bloom were less genetically differentiated than samples from the end of the bloom events. These last samples formed distinct clusters according to their geographic origin (Fig. 2; cf.

PZ11C, PZ11D compared to RC11D and RC11C).

Genetic differentiation between pairs of samples (F st ) ranged from -0.0035 (RC11A vs. RC11B) to 0.0770 (RC10A vs. PZ11C), whereas D est ranged from 0.0589 (RC11B vs. RC11C) to 0.4695 (RC10A vs. PZ11C) (Table 3S). The lowest F st values were observed for two samples from the same site in the same year at two consecutive in-bloom sampling dates, whereas the highest values were observed for samples between different sites and different years. The tests of differentiation at different spatial and temporal levels confirm the PCA results. They indicate that the early bloom samples in Penzé and Rance taken in 2010 (PZ10A and RC10A) differed significantly from all other samples (Table S3).

The first AMOVA helped gauge the importance of spatial variation with respect to phase variation within a bloom. This AMOVA could only be applied on the 2011 samples (Table 4A) and revealed that the genetic differentiation was significant between sites and among bloom phases within sites. More specifically, the percent of variation explained by the genetic differentiation during the different phases of the bloom event was three times higher (3.87%) than the genetic differentiation between the Penzé and Rance sites (1.23%, Table 4A).

The second AMOVA was used to analyze the importance of temporal variation at two hierarchical levels: interannual and intrabloom (Table 4B). This temporal AMOVA could only be applied to the Penzé estuary. The results show again that the two levels of differentiation were significant. The percent variation explained by genetic differentiation between the different in-bloom periods was weaker (4.27%, Table 4B) than that explained by genetic differentiation between 2010 and 2011 (10.01%, Table 4B).

Discussion

Clonal reproduction versus sexual reproduction

The reproductive system can play an important role in the functioning and evolution of populations by modifying their dynamics, their diversity and their population genetic structure. As in many other phytoplankton species, the dinoflagellate species A. minutum is characterized by high rates of clonal reproduction during the development of bloom events, followed by a period of sexual reproduction that produces resting cysts, coinciding with the maximum and the decline of cell concentrations, respectively [START_REF] Garcés | Relationship between vegetative cells and cyst production during Alexandrium minutum bloom in Arenys de Mar harbour (NW Mediterranean)[END_REF][START_REF] Bravo | The intricacies of dinoflagellate pellicle cysts: The example of Alexandrium minutum cysts from a bloomrecurrent area (Bay of Baiona, NW Spain)[END_REF]. During periods of asexual division, differences in selective values between clonal lines can rapidly lead to differences in genotype frequencies. Numerical simulations show that selective sweeps can occur when rapidly growing clones become more abundant within a population [START_REF] Rynearson | DNA fingerprinting reveals extensive genetic diversity in a field population of the centric diatom Ditylum brightwellii[END_REF]. During a bloom event, advantageous genotypes may increase in frequency, leading to an overall decrease in genetic diversity via genetic hitchhiking over the whole genome (Maynard- [START_REF] 'rourke | How clonal are bacteria[END_REF]. Even in the absence of selection, low clonal diversity and linkage equilibrium provide evidence that sexual reproduction occurs in a population with clonal reproduction. At the end of a bloom, the most frequent clones would thus preferentially participate in the production of the following generation via sexual reproduction. However, in rapidly growing organisms with short generation times, the huge population sizes can be considered as virtually infinite and genotypic diversity can thus be maintained during the bloom events, even if organisms primarily reproduce clonally [START_REF] Bengtsson | Genetic variation in organisms with sexual and asexual reproduction[END_REF].

In this study, all the analyzed clones were genetically distinct, suggesting that bloom events involve huge effective population sizes. The high genetic diversity observed here indicates that asexual reproduction does not predominate within A. minutum populations. It however does suggest that blooms were initiated by numerous different sexual cysts. Our results corroborate the recent observations reported for Alexandrium fundyense showing unique multilocus genotypic frequencies of 81% to 97% [START_REF] Erdner | Diversity and dynamics of a widespread bloom of the toxic dinoflagellate Alexandrium fundyense[END_REF][START_REF] Richlen | Extensive genetic diversity and rapid population differentiation during blooms of Alexandrium fundyense (Dinophyceae) in an isolated salt pond on Cape Cod, MA, USA[END_REF]. Likewise, a study on Alexandrium tamarense has shown that the probability of sampling clones with the same genotype is practically nil due to the large population sizes of blooms [START_REF] Nagai | Microsatellite markers reveal population genetic structure of the toxic dinoflagellate Alexandrium tamarense (Dinophyceae) in japanese coastal waters[END_REF]. The high genotypic diversity as well as the high gene diversity values observed in our study (0.78 < H s < 0.87) are comparable to those of Mediterranean populations of the same species (0.62 < H s < 0.89; [START_REF] Casabianca | Population genetic structure and connectivity of the harmful dinoflagellate Alexandrium minutum in the Mediterranean Sea[END_REF]. There are reports of similar high genotypic diversity values in other phytoplankton species, such as the haptophyte (Prymnesium parvum) and the Raphidophyceae species (Gonyostomum semen) using AFLP genetic markers [START_REF] Barreto | AFLP fingerprinting shows that a single Prymnesium parvum harmful algal bloom consists of multiple clones[END_REF][START_REF] Lebret | Genetic diversity within and genetic differentiation between blooms of a microalgal species[END_REF].

The continuous germination of resting cysts during the entire bloom event [START_REF] Lebret | Genetic diversity within and genetic differentiation between blooms of a microalgal species[END_REF], as well as the specific cohort of these cysts produced during successive annual blooms and stored in the cyst bank, may enhance genetic diversity (in terms of genotypes and genes) [START_REF] Alpermann | Implications of lifehistory transitions on the population genetic structure of the toxigenic marine dinoflagellate Alexandrium tamarense[END_REF]. Furthermore, we cannot exclude the possibility that sexual reproduction maintains genetic diversity, even during a bloom event. Observations in in vitro cultures suggest that the zygote (planozygote) may undergo meiosis without forming a resting cyst [START_REF] Figueroa | Comparative study of the life cycles of Alexandrium tamutum and Alexandrium minutum (Gonyaulacales, Dinophyceae) in culture[END_REF], thereby producing new vegetative cells. This type of reproduction may be promoted by the presence of the parasite Parvilucifera sinerae (Alveolata), and shortcircuiting the resting cyst stage may possibly constitute a strategy to avoid the parasite [START_REF] Figueroa | Reproductive plasticity and local adaptation in the host parasite system formed by the toxic Alexandrium minutum and the dinoflagellate parasite Parvilucifera sinerae[END_REF]. Two different species belonging to genus Parvilucifera were detected during the 2010 and 2011 sampling cruises (Lepelletier et al. submitted). However, unfortunately, there have been no investigations to identify this type of reproduction.

In our study, no global multilocus linkage disequilibrium was detected, suggesting that recombination is sufficient to break down linkages between loci, confirming that A. minutum reproduces sexually. Only one sample taken during the late bloom period in Penzé in 2011 (PZ11D) showed significant multilocus linkage disequilibrium, as estimated using r̅ d ).

Spatiotemporal genetic structure of bloom events

The results of the microsatellite analysis clearly indicate that A. minutum blooms are structured both in space and in time. Our study shows that there is a temporal genetic differentiation during and between A. minutum blooms as well as spatial differentiation between sites. Thus, the overall genetic differentiation observed between the Rance and Penzé estuaries separated by roughly 150 km was significant. This result suggests that gene flow via tidal currents is limited in A. minutum, as demonstrated in A. tamarense [START_REF] Nagai | Microsatellite markers reveal population genetic structure of the toxic dinoflagellate Alexandrium tamarense (Dinophyceae) in japanese coastal waters[END_REF]).

Given the short duration of A. minutum bloom events (30 days on average) and that they are geographically restricted to estuaries, the gene flow between populations is likely limited.

Genetic differences observed between the two estuaries late bloom samples confirm the genetic isolation between estuaries during the bloom period. However, early bloom samples showed weaker spatial differentiation (Table 4). This observation suggests that the Penzé and Rance estuaries communicate during spring tides, potentially transporting vegetative cells or cysts via the residual west-east tidal currents. During the bloom season, which preferentially occurs during neap tides, the two estuaries are likely isolated and the accumulation of bloom biomass is mainly due to a response to water flow and to constant, suitable environmental conditions (i.e. salinity temperature, irradiance and nutrient supply) [START_REF] Anderson | The globally distributed genus Alexandrium: Multifaceted roles in marine ecosystems and impacts on human health[END_REF]). In our study, interannual genetic differentiation was greater than intrabloom differentiation, suggesting that blooms are not only made up of newly formed cysts produced during the preceding year, but also of recruitment from migrant vegetative cells and/or from an older cyst bank [START_REF] Estrada | The role of resting cysts in Alexandrium minutum population dynamics[END_REF]. Even if a multigenerational cyst bank gives rise to a bloom event, the germination of cysts may be influenced by environment conditions and, by chance or selection, samples become more differentiated than the more homogeneous cyst bank that produced them [START_REF] Mccue | Seed bank influences on genetic diversity in the rare annual Clarkia springvillensis (Onagraceae)[END_REF]. The constant germination of the cyst bank may thus lead to a decrease in the interannual F st . Nevertheless, the cyst bank does not appear to contribute regularly to bloom events because the 2010 Penzé sample PZ10D was not genetically different from the early 2011 Penzé bloom sample (PZ11A). Therefore, the 2011 early bloom sample may have been composed at least partly of cysts produced in the preceding year.

In isolated estuaries, there were significant genetic changes during a bloom event (see AMOVA, Table 4A). Although no additional signatures of selection, such as multilocus linkage disequilibrium, could be detected with the neutral markers used in this study, we believe that, in these very large populations, genetic changes may nevertheless be driven by selection (see below). These genetic changes are concordant with those reported for A. fundyense blooms in Massachusetts where temporal intrabloom differentiation has also been observed [START_REF] Richlen | Extensive genetic diversity and rapid population differentiation during blooms of Alexandrium fundyense (Dinophyceae) in an isolated salt pond on Cape Cod, MA, USA[END_REF]. Two factors can account for this temporal differentiation.

First, changes may be due to delayed germination of different cohorts of cysts buried in the sediments and/or continuous encystment by a small percentage of motile cells [START_REF] Pitcher | The dinoflagellate Alexandrium minutum in Cape Town harbour (South Africa): Bloom characteristics, phylogenetic analysis and toxin composition[END_REF]. In Gonyostomum semen (Raphidophyceae), [START_REF] Lebret | Genetic diversity within and genetic differentiation between blooms of a microalgal species[END_REF] suggest that continuous germination of cysts during a bloom is a key element behind rapid genetic changes. Second, these changes may be the result of local abiotic or biotic selection as suggested for the diatom Ditylum brightwellii [START_REF] Rynearson | Maintenance of clonal diversity during a spring bloom of the centric diatom Ditylum brightwellii[END_REF][START_REF] Rynearson | Spring bloom development, genetic variation, and population succession in the planktonic diatom Ditylum brightwellii[END_REF]. In these large populations, the effects of natural selection may predominate over those of genetic drift on genetic structure. Thus, in D. brightwellii, the succession of genetically distinct populations in different environmental conditions appears to reflect a tight coupling between environment and genetics [START_REF] Rynearson | Spring bloom development, genetic variation, and population succession in the planktonic diatom Ditylum brightwellii[END_REF]. Of the various abiotic factors, seasonal temperature variation is one parameter that can explain the maintenance of genetic diversity (Maynard- [START_REF] Smith | Polymorphism in a varied environment: how robust are the models[END_REF][START_REF] Bell | The masterpiece of nature: the evolution and genetics of sexuality Springer[END_REF]. Recent studies on the diatom Asterionella formosa have demonstrated that variation in temperature may contribute to maintaining genetic diversity in blooms [START_REF] Gsell | Genotype-bytemperature interactions may help to maintain clonal diversity in Asterionella formosa (Bacillariophyceae)[END_REF]. In this species, reaction norms of clone growth studied as a function of environmental temperature reveal considerable genetic diversity within a bloom. In our study, fluctuating temperatures may explain not only the maintenance of genetic diversity, but also genetic changes during a bloom event. However, biotic factors, such as predation and/or parasitism, may also play a role in determining population structure and dynamics [START_REF] Montagnes | Responsibility of microzooplankton and parasite pressure for the demise of toxic dinoflagellate blooms[END_REF]. In A. minutum, eukaryotic parasites such as Parvilucifera spp. and Amoebophrya spp. can regulate blooms [START_REF] Chambouvet | Control of toxic marine dinoflagellate blooms by serial parasitic killers[END_REF][START_REF] Figueroa | Reproductive plasticity and local adaptation in the host parasite system formed by the toxic Alexandrium minutum and the dinoflagellate parasite Parvilucifera sinerae[END_REF]Lepelletier et al. submitted). The impact of parasitism can be tested by analyzing the fitness of different strains and their resistance to parasites according to date and sampling site. Whatever the factors involved, our study illustrates how sexual reproduction and/or migration are likely to erase any selective effects that arise each year during the vegetative phase of a bloom event. Table 1: Characteristics of the spatiotemporal sampling. Sample sizes (N) are given for strains that were isolated for culturing and for the surviving strains that were genotyped. Codes for samples: PZ for Penzé, RC for Rance; 10 for 2010 and 11 for 2011; A, B, C and D indicate respectively the different periods of the blooms: the beginning, the middle 1, the middle 2 and the end of the bloom. Cells were counted in a counting chamber using an inverted microscope (Olympus CKX41).

Figure legends:

Figure 1 :

 1 Figure 1: Maps of the study area showing sampling stations in the two estuaries: Penzé (PZ)

Figure 2 :

 2 Figure 2: PCA of genetic differentiation between samples taken from the Penzé and Rance

Figure S1 .

 S1 Figure S1. Quantification of DNA in Alexandrium minutum cultures in comparison with

Figure S2 .

 S2 Figure S2. Resolving power of the microsatellite marker set to discriminate the maximum

Table 2 :

 2 Genetic diversity estimates in A. minutum at each of the 11 sampling sites and dates and for each of the seven microsatellite loci. N, number of individuals genotyped in each sample; N a , number of alleles per locus; R a , expected allelic richness based on the smallest sample size (10); H s , Nei's gene diversity.

	Site/Year	Sampling	Sample code	Cells density	Temperature of	Number of	Number of	Number of	Number of
		date		x 10 3 cells/L	water (air	strains	surviving	A. minutum	strains
					temperature min./	isolated	strains (%)	strains after	genotyped
					max.) (°C)			screening
	Penzé/2010	02 June	PZ10A	8	14.5 (8.6/22)	82	36 (43.9)	32	24
		30 June	PZ10D	102	19.0 (13.5/24.5)	29	16 (55.2)	09	09
		01 July	PZ10D	63	19.3 (12.2/25.9)	55	30 (54.5)	14	08
	Penzé/2011	01 June	PZ11A	31	17.9 (2.7/18.6)	85	55 (64.7)	22	19
		15 June	PZ11B	88	18.0 (12.3/20.5)	93	46 (49.4)	24	14
		17 June	PZ11B	171	15.9 (8.4/16.3)	144	36 (25)	24	15
		22 June	PZ11C	402	16.0 (11.5/18)	119	49 (41.2)	32	24
		11 July	PZ11D	ND	17.5 (9/23.8)	195	30 (15.4)	26	18
	Rance/2010	02 June	RC10A	36	16.3 (11.2/17.8)	65	43 (66.2)	43	40
	Rance/2011	09 May	RC11A	13	15.4 (9.8/19.7)	45	26 (57.8)	19	15
		23 May	RC11B	168	16.8 (7.4/24.2)	96	55 (55.2)	40	25
		27 May	RC11C	288	17.0 (10.8/15.1)	96	57 (59.3)	38	25
		03 June	RC11D	66	18.5 (13/22.1)	83	22 (26.5)	17	10
		05 June	RC11D	106	18.8 (13.8/14.7)	53	29 (54.7)	24	19
					Total	1240	530 (42.7)	364

Table 3 :

 3 Multilocus measurements of linkage disequilibrium using r̅ d[START_REF] Agapow | Indices of multilocus linkage disequilibrium[END_REF].

	Sample	PZ10A	PZ10D	PZ11A	PZ11B	PZ11C	PZ11D	RC10A	RC11A	RC11B	RC11C	RC11D
	(N)	(24)	(17)	(19)	(29)	(24)	(18)	(40)	(15)	(25)	(25)	(29)
	r̅ d	-0.008 0.068	-0.034 -0.017 0.037	0.005	0.003	0.027	-0.037	-0.049	0.009
	P-value	0.566	0.047	0.870	0.730	0.063	0.416	0.423	0.240	0.902	0.971	0.348
	Table 4: Spatial (A) and temporal (B) analyses of bloom events
	A: Spatial AMOVA: Variation among bloom phases between the Penzé and Rance sites
	sampled in 2011 (F st = 0.05. P< 10 -5 )
	Source of variation		df	Sum of	Variance	Percentage	P-value
						squares	component	variation (%)
	Between the Penzé and Rance	1	13.91	0.04		1.23		0.008
	sites sampled in 2011
	Among bloom phases within	6	44.51	0.12		3.87		0.002
	site
	Within bloom phase		318	896.08	2.82		94.88
	Total					954.49	2.97
	B: Temporal AMOVA: Variation among bloom phases between years 2010 and 2011 at
	Penzé site (F st = 0.14. p< 10 -5 )
	Source of variation		df	Sum of	Variance	Percentage	P-value
						squares	component	variation (%)
	Between years 2010 and 2011	1	41.24	0. 33		10.01	p< 10 -5
	in Penzé
	Among bloom phases within	4	33.04	0.14		4.27		p< 10 -5
	year
	Within bloom phase		226	648.12	2.86		85. 72
	Total					722.41	3.34

Table S2 .

 S2 Microsatellite data amplified on 265 individual strains at seven loci. Population codes are given in Table1.

	PZ522 PZ966 PZ658 RC925 RC1034	PZ10D PZ11B PZ11D RC11A RC11C	199 197 0 200 197	200 202 162 200 218	240 248 238 239 228	263 273 263 259 275	187 0 0 196 202	240 0 260 231 226	216 198 237 208 202
	PZ579 PZ967 PZ683 RC931 RC1040	PZ11A PZ11B PZ11D RC11A RC11C	182 0 197 197 229	200 0 210 202 0	228 0 229 239 230	0 274 274 255 0	204 208 204 206 202	239 240 240 240 233	0 208 185 191 189
	PZ594 PZ968 RC275 RC937 RC1042	PZ11A PZ11B RC10A RC11A RC11C	197 192 0 197 197	202 202 0 204 200	228 252 208 248 237	248 269 279 261 255	199 194 214 201 197	234 248 214 240 233	194 189 0 189 189
	Individuals PZ582 PZ969 RC277 RC938 RC1043 PZ591 PZ970 RC280 RC939 RC1044	PZ11A PZ11B RC10A RC11A RC11C Sample codes PZ11A PZ11B RC10A RC11A RC11C	197 196 195 197 197 Aminu22 Aminu41 Aminu11 Aminu20 Aminu43 Aminu48 200 228 273 202 239 210 229 279 200 0 200 229 261 202 239 202 242 269 200 243 200 239 261 0 242 197 202 237 0 194 239 200 200 229 279 0 246 207 202 238 237 175 235 195 200 238 261 193 240 200 200 237 255 200 247	190 189 192 184 187 Aminu08 187 189 184 206 191
	PZ374 PZ584 PZ788 RC281 RC919 RC1047	PZ10A PZ11A PZ11C RC10A RC11A RC11C	199 197 200 197 197 197	202 202 200 200 202 0	255 257 236 223 236 248	0 259 0 0 259 251	201 196 198 202 198 202	215 239 239 241 240 238	185 185 190 175 185 176
	PZ352 PZ593 PZ770 RC283 RC921 RC1049	PZ10A PZ11A PZ11C RC10A RC11A RC11C	199 197 199 197 0 0	200 200 200 204 200 180	228 248 248 229 253 237	279 0 258 255 248 263	201 202 188 202 203 196	0 237 240 250 237 238	190 179 187 189 187 187
	PZ330 PZ587 PZ753 RC297 RC926 RC1050	PZ10A PZ11A PZ11C RC10A RC11A RC11C	197 199 199 197 0 197	164 202 202 215 200 0	240 236 228 249 250 229	248 0 253 271 248 269	0 201 205 202 205 200	0 239 242 211 239 242	180 190 190 213 0 164
	PZ350 PZ589 PZ761 RC304 RC927 RC1055	PZ10A PZ11A PZ11C RC10A RC11A RC11C	197 199 197 182 197 197	202 202 202 226 200 0	228 259 257 251 237 229	279 261 263 0 0 276	0 209 201 0 205 200	176 244 237 231 239 238	185 185 201 213 0 216
	PZ344 PZ583 PZ764 RC322 RC930 RC1059	PZ10A PZ11A PZ11C RC10A RC11A RC11C	197 199 195 183 197 197	200 200 202 199 200 197	228 242 0 224 238 255	0 0 264 259 0 0	215 207 203 175 205 204	217 239 240 0 240 240	185 185 185 187 172 216
	PZ341 PZ572 PZ789 RC317 RC932 RC1061	PZ10A PZ11A PZ11C RC10A RC11A RC11C	197 197 195 202 199 215	232 200 200 202 0 0	248 257 259 238 0 239	248 261 0 0 0 0	230 204 198 0 203 0	234 256 242 176 240 244	185 234 187 205 213 175
	PZ362 PZ575 PZ754 RC307 RC934 RC1062	PZ10A PZ11A PZ11C RC10A RC11A RC11C	197 0 190 197 0 197	200 0 200 200 0 0	238 240 236 238 238 239	279 0 267 248 248 265	188 202 204 0 203 193	0 235 239 0 240 218	190 187 174 0 210 187
	PZ339 PZ577 PZ757 RC321 RC1003 RC1035	PZ10A PZ11A PZ11C RC10A RC11B RC11C	197 0 195 197 197 195	232 206 200 204 200 204	0 0 237 236 248 252	0 265 259 0 253 0	202 171 191 194 202 197	0 242 239 0 0 242	190 186 189 0 188 185
	PZ351 PZ578 PZ758 RC271 RC1013 RC1036	PZ10A PZ11A PZ11C RC10A RC11B RC11C	197 0 195 197 197 183	202 0 200 200 200 210	236 239 226 248 239 236	250 0 269 279 268 261	197 206 204 188 204 0	214 239 242 176 238 242	190 217 184 0 187 172
	PZ359 PZ580 PZ759 RC293 RC1014 RC1056	PZ10A PZ11A PZ11C RC10A RC11B RC11C	197 197 199 197 0 206	162 202 202 210 202 200	240 242 242 242 239 253	279 253 263 261 271 253	188 197 0 198 200 205	0 236 0 240 240 242	199 225 0 0 225 0
	PZ331 PZ581 PZ762 RC323 RC1016 RC1039	PZ10A PZ11A PZ11C RC10A RC11B RC11C	195 197 195 197 199 197	202 202 202 200 200 202	236 237 229 0 0 242	0 237 267 279 258 253	205 202 208 180 200 207	240 240 239 217 239 239	187 189 187 180 187 187
	PZ360 PZ586 PZ766 RC301 RC974 RC1060	PZ10A PZ11A PZ11C RC10A RC11B RC11C	179 0 199 197 197 195	0 202 200 200 202 202	236 259 242 278 236 252	279 261 269 0 0 258	221 208 202 0 200 202	0 244 239 226 240 240	190 189 187 185 224 187
	PZ353 PZ590 PZ767 RC316 RC975 RC854	PZ10A PZ11A PZ11C RC10A RC11B RC11D	0 0 0 197 197 197	218 155 202 202 200 202	0 240 0 244 238 248	279 0 265 0 265 0	220 0 212 0 198 0	0 229 239 214 328 242	185 187 184 187 187 187
	PZ345 PZ355 PZ595 PZ771 RC311 RC978 RC855 PZ356 PZ597 PZ773 RC310 RC979 RC857 PZ340 PZ689 PZ775 RC315 RC980 RC858 PZ333 PZ691 PZ777 RC296 RC981 RC859 PZ334 PZ696 PZ778 RC325 RC982 RC860 PZ337 PZ338 PZ348 PZ354 PZ369 PZ482 PZ484 PZ697 PZ701 PZ704 PZ708 PZ711 PZ712 PZ780 PZ781 PZ782 PZ784 PZ785 RC327 RC295 RC983 RC861 RC298 RC984 RC863 RC305 RC986 RC864 RC313 RC987 RC867 RC308 RC988 RC873 PZ714 PZ787 RC303 RC992 RC874 PZ665 RC314 RC999 RC875 PZ485 PZ715 PZ661 RC324 RC1008 RC876 PZ487 PZ716 PZ645 RC328 RC1001 RC877 PZ488 PZ719 PZ648 RC287 RC1005 RC878 PZ489 PZ721 PZ682 RC300 RC1015 RC880 PZ491 PZ742 PZ643 RC282 RC994 RC881 PZ492 PZ493 PZ520 PZ521 PZ524 PZ514 PZ509 PZ500 PZ516 PZ647 RC1032 PZ743 PZ749 PZ959 PZ960 PZ961 PZ962 PZ963 PZ964 PZ965 PZ679 PZ660 RC302 RC991 RC884 PZ655 RC294 RC1011 RC885 PZ637 RC273 RC1019 RC889 PZ639 RC292 RC1020 RC894 PZ640 RC320 RC1021 RC895 PZ641 RC326 RC1022 RC897 PZ644 RC913 RC1024 RC898 PZ646 RC915 RC1026 RC883 RC922 RC1028 RC886	PZ10A PZ10A PZ11A PZ11C RC10A RC11B RC11D PZ10A PZ11A PZ11C RC10A RC11B RC11D PZ10A PZ11B PZ11C RC10A RC11B RC11D PZ10A PZ11B PZ11C RC10A RC11B RC11D PZ10A PZ11B PZ11C RC10A RC11B RC11D PZ10A PZ10A PZ10A PZ10A PZ10A PZ10D PZ10D PZ11B PZ11B PZ11B PZ11B PZ11B PZ11B PZ11C PZ11C PZ11C PZ11C PZ11C RC10A RC10A RC11B RC11D RC10A RC11B RC11D RC10A RC11B RC11D RC10A RC11B RC11D RC10A RC11B RC11D PZ11B PZ11C RC10A RC11B RC11D PZ11D RC10A RC11B RC11D PZ10D PZ11B PZ11D RC10A RC11B RC11D PZ10D PZ11B PZ11D RC10A RC11B RC11D PZ10D PZ11B PZ11D RC10A RC11B RC11D PZ10D PZ11B PZ11D RC10A RC11B RC11D PZ10D PZ11B PZ11D RC10A RC11B RC11D PZ10D PZ10D PZ10D PZ10D PZ10D PZ10D PZ10D PZ10D PZ10D PZ11D RC11C PZ11B PZ11B PZ11B PZ11B PZ11B PZ11B PZ11B PZ11B PZ11B PZ11D PZ11D RC10A RC11B RC11D PZ11D RC10A RC11B RC11D PZ11D RC10A RC11C RC11D PZ11D RC10A RC11C RC11D PZ11D RC10A RC11C RC11D PZ11D RC10A RC11C RC11D PZ11D RC11A RC11C RC11D PZ11D RC11A RC11C RC11D RC11A RC11C RC11D	0 0 210 199 197 199 197 0 197 197 197 200 197 0 197 195 197 197 0 195 197 199 197 197 196 197 194 197 197 195 198 197 199 199 197 197 201 197 197 195 195 199 197 0 204 195 199 197 199 195 195 197 197 195 197 195 195 197 197 195 199 197 195 195 197 195 199 193 197 195 199 193 197 207 199 195 199 193 197 202 199 197 199 190 197 197 199 197 197 182 197 200 0 197 197 182 191 197 197 197 197 182 188 197 197 168 199 197 195 195 182 179 0 207 0 199 212 197 197 193 195 197 197 0 197 183 156 180 195 0 0 0 197 197 0 194 0 198 0 0 197 199 0 197 196 197 0 197 196 199 195 197 195 197 193 197 197 193 0 197	0 232 200 202 202 202 224 207 200 200 200 200 200 163 202 204 202 200 0 202 200 200 200 200 0 200 202 202 200 200 202 202 202 204 206 202 200 204 200 202 200 200 0 176 200 200 202 200 202 202 202 202 0 200 200 202 202 210 202 200 202 0 0 196 200 196 202 200 200 200 210 0 202 202 204 202 202 204 200 202 200 202 202 200 162 202 204 202 200 200 200 201 0 200 202 0 200 202 204 200 202 181 0 202 200 202 200 200 202 202 202 202 200 200 212 202 202 196 200 202 200 202 232 223 200 200 218 0 202 202 202 208 202 200 210 223 204 0 200 200 200 200 223 202 200 202 210 202 200 200 200 202 200 200 0 196 202 202 200 202	287 248 236 229 248 229 241 238 0 229 0 229 237 248 239 229 0 238 243 237 229 242 240 263 229 238 259 229 278 245 255 255 239 242 229 267 244 229 237 239 257 229 242 242 236 228 238 229 244 240 228 239 255 248 239 239 238 244 237 248 240 239 0 229 257 229 239 0 237 248 242 0 237 259 225 248 253 238 238 0 225 248 228 238 244 251 225 239 228 0 240 237 225 239 252 238 0 238 239 239 244 248 248 238 249 240 242 252 242 258 242 240 0 242 224 229 237 248 229 257 223 238 227 244 228 248 240 229 257 0 236 255 237 239 278 239 243 237 223 242 229 255 248 236 239 242 223 257 243 239 244 237 237 248 229 237 236 229 198 236	0 279 265 0 248 263 260 279 0 273 246 255 265 279 269 259 0 253 0 236 267 257 248 278 267 253 259 261 279 269 261 273 269 265 267 247 271 261 270 274 261 261 263 259 0 261 270 0 270 279 248 261 261 0 261 267 0 269 285 248 252 267 0 0 0 265 271 0 259 267 248 0 261 261 263 253 248 279 258 0 0 283 248 279 0 259 248 253 248 265 258 253 231 264 0 248 0 238 261 262 270 279 259 271 251 0 260 262 248 0 261 261 279 271 0 0 261 261 265 257 248 248 271 265 254 248 0 269 267 248 248 273 261 248 279 0 261 269 0 0 251 253 0 259 262 257 0 267 262 269 261 261 261 261 0 273 0 259 261 0	188 221 204 200 188 201 204 197 198 198 188 0 0 188 0 197 0 0 202 200 196 208 0 195 202 204 198 206 184 200 196 189 0 0 187 0 197 204 200 196 197 197 198 204 200 208 204 198 198 221 0 202 196 0 207 201 198 202 0 0 198 0 0 191 0 212 204 201 0 199 209 217 209 204 200 206 192 188 203 204 211 204 207 180 205 191 200 204 197 213 198 0 0 201 203 221 218 0 197 202 200 0 194 208 200 187 190 202 205 197 198 206 206 0 196 200 204 206 202 210 0 204 0 202 207 0 203 197 204 0 196 203 0 200 188 0 201 199 201 0 193 200 188 198 193 204 220 202 201 200 0 200 0 204 200 0 205 196 204 199	252 176 234 239 176 0 240 214 241 233 180 0 238 176 251 231 240 240 235 239 239 241 214 240 0 239 239 241 176 242 240 240 242 240 238 206 239 0 242 238 239 239 241 241 241 239 242 256 240 176 226 242 240 240 0 240 181 240 240 214 240 257 240 236 240 210 242 181 240 240 242 214 240 240 231 218 239 176 239 0 232 240 237 176 239 240 234 0 239 176 244 264 210 240 239 236 230 242 248 240 239 176 239 237 0 228 240 242 239 239 225 218 229 239 238 240 239 0 246 240 246 240 240 240 240 239 226 0 240 236 214 239 240 229 176 242 228 239 214 258 240 229 176 242 240 242 176 240 242 239 237 242 240 229 240 240 239 240 240 240	185 0 189 199 0 189 190 191 189 205 216 194 190 179 192 205 189 195 197 210 225 213 187 180 187 0 198 230 187 0 189 191 0 189 0 174 179 187 189 187 226 184 225 189 189 189 187 0 187 190 0 0 190 0 197 185 185 184 187 205 216 187 205 197 184 187 187 185 179 0 190 205 180 184 0 187 205 172 190 216 200 190 185 185 187 226 187 0 0 190 190 216 201 0 0 230 227 0 190 190 199 230 187 0 187 220 185 194 185 190 190 187 187 184 199 189 187 189 189 189 187 211 187 184 207 0 187 187 227 0 185 222 190 190 199 179 233 197 187 187 229 199 184 187 211 205 216 185 221 189 201 185 189 221 206 190 0 185 195 190

Table S3 :

 S3 Pairwise differentiation between the 11 populations of Alexandrium minitum. F st (θ) values are given above diagonal and Jost's D est below diagonal. Significant F st values (1000 permutations) are depicted in bold. *: F st values that remain significant after a Bonferroni correction.Population codes are given in Table1.

		PZ10A	PZ10D	PZ11A	PZ11B	PZ11C	PZ11D	RC10A	RC11A	RC11B	RC11C	RC11D
	PZ10A		0.0396	0.0265	0.0378* 0.0639* 0.0403	0.0161	0.0577	0.0456* 0.0443* 0.0486*
	PZ10D	0.3228		0.0124	0.0247	0.0170	0.0122	0.0495* 0.0281	0.0234	0.0254	0.0495*
	PZ11A	0.1847	0.2007		0.0073	0.0211	0.0019	0.0536* 0.0331	0.0100	0.0104	0.0236
	PZ11B	0.2748	0.2279	0.0929		0.0102	0.0234	0.0522* 0.0062	0.0008	0.0059	0.0103
	PZ11C	0.3810	0.2206	0.1694	0.0576		0.0259	0.0770* 0.0401	0.0260	0.0429* 0.0534*
	PZ11D	0.2135	0.2096	0.0956	0.2102	0.1968		0.0623* 0.0318	0.0197	0.0366	0.0581*
	RC10A	0.1365	0.4011	0.3541	0.3287	0.4695	0.3464		0.0457	0.0544* 0.0582* 0.0763*
	RC11A	0.2989	0.2938	0.2065	0.1187	0.2487	0.2071	0.2295		-0.0035	0.0259	0.0179
	RC11B	0.2696	0.2414	0.1631	0.0766	0.1448	0.1700	0.3007	0.0684		-0.0011	0.0163
	RC11C	0.3100	0.2402	0.1157	0.0994	0.2331	0.2323	0.3723	0.2081	0.0589		0.0169
	RC11D	0.2225	0.3240	0.1177	0.1145	0.2802	0.2610	0.3815	0.1453	0.1225	0.1156

Acknowledgements

We are very grateful to Yannis Michalakis, Denis Roze and Carolyn Engel for constructive comments on an earlier draft and 3 anonymous reviewers who helped improve this manuscript. We warmly thank the IFREMER laboratory in Dinard (France) and especially Claude Lebec and Claire Rollet. Thanks to the Roscoff Culture Collection (RCC), Service Mer et Observation (Marine Operations Department) at the Roscoff Biological Station and all the partners in the Paralex project for their help with sampling. Sequences and microsatellite genotypes were generated by Morgan Perennou and Gwenn Tanguy at the Roscoff Biological Station (Ouest-Génopole). This project was carried out in partial fulfillment of Aliou Dia's PhD degree, supported by funds from the ANR and a PhD fellowship from the Brittany Regional Council (ARE10087: POPALEX). This work was financially supported by the Brittany Regional Council, the CNRS program EC2CO, the French ANR projects PARALEX ("The sixth extinction" ANR-2009-PEXT-01201) and CLONIX ("Programme Blanc" ANR11-BSV7-00704), the European Project MaCuMBa (FP7-KBBE-2012-6-311975) and the EC2CO project PALMITO.

Data accessibility

Data used in this study (supplementary Table S1) have been deposited at the Roscoff Culture Collection (www.sb-roscoff.fr/Phyto/RCC).

Authors' contributions

AD conceived the study, performed isolation and culture of strains, molecular data, analysis and drafted the manuscript, SM contributed to molecular analyses; EB performed isolation and culture of strains, DM performed cytometry analyses, MV participated in the design of the study, performed some statistical analysis and revised the manuscript, LG and CD coordinated the study, helped draft and revised the manuscript. All authors contributed to sample collection, read and approved the final manuscript.

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SPATIOTEMPORAL CHANGES IN THE GENETIC DIVERSITY OF HARMFUL ALGAL BLOOMS CAUSED BY THE TOXIC DINOFLAGELLATE ALEXANDRIUM MINUTUM


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Christoph Grunau

Envi- Ronmentally

Environmentally induced epigenetic plasticity in the human parasite Schistosoma mansoni

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

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Immuninv 2014

Dijon

David Mai

Duval

Richard Galinier

Gabriel Mouahid

Eve Toulza

Anne Rognon

Nathalie Arancibia

Jean Francois Allienne

Guillaume Mitta

André Théron

Benjamin Gourbal

Paenibacillus, a novel pathogen of Biomphalaria glabrata, an intermediate host for schistosomiasis

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Schistosomiasis is the second most widespread tropical parasitic disease after malaria. To achieve the objective of schistosomiasis eradication in a decade, various research strategies and treatment programs were recommended and supported by WHO. One of these applicable approaches is based on the control of snail vectors in endemic area. Previous field studies have shown that competitor or predator introduction could be effective but no systemic investigation has ever been conducted to identify snail microbial pathogen and evaluate its molluscicide effect. In this work, our aim is to provide a characterization of a novel infectious agent isolated from unhealthy Biomphalaria snails in our laboratory.

Unhealthy snails exhibits white nodules.

Discussion

Paenibacillus sp closely related to P. alvei has been isolated from unhealthy Biomphalaria snails. Histopathological examination has shown massive bacterial infiltration leading to an overall disorganization of snail tissues. Exposure of healthy snails to Paenibacillus infected snails led to a massive mortality. Moreover, egg hatching was significantly lower in exposed snails than in control whereas the spawning appeared to be unaffected. Embryonic lethality is correlated with the presence of this pathogenic bacteria in eggs. This study reports the first description of a novel Paenibacillus strain as snail microbial pathogen by affecting both adult and embryonic stages of Biomphalaria, the vector snail of schistosomiasis.

1 Université Perpignan Via Domitia, Ecologie et Evolution des Interactions, UMR 5244, F-66860,Perpignan, France .

2 CNRS, Ecologie et Evolution des Interactions,UMR 5244, F-66860, Perpignan, France.

Dissected snail presents nodules on mantle (Mt), hepato-pancreas (Hp) and ovotestis (Ov) region. These rod-shaped bacteria present a mixed pattern of Gram staining and produce some endospores.

Massive bacterial infiltration in snail tissues

  Paenibacillus, a novel pathogen of Biomphalaria glabrata, an intermediate host for schistosomiasis David Duval, Richard Galinier, Gabriel Mouahid, Eve Toulza, Anne Rognon, Nathalie Arancibia, Jean-François Allienne, Guillaume Mitta, André Théron, Benjamin Gourbal To cite this version: David Duval, Richard Galinier, Gabriel Mouahid, Eve Toulza, Anne Rognon, et al.. Paenibacillus, a novel pathogen of Biomphalaria glabrata, an intermediate host for schistosomiasis. ImmunInv 2014 (Colloque Immunité des Invertébrés), May 2014, Dijon, France. . hal-01134490

  the headfoot (A) and the kidney (B) regions of infected snails. BC: Bacterial colonies; BS: Blood space; DCT: Dense connective tissue ; EC: Epithelial cells; L: Lumen; MC: Mantle cavity; PV: Pulmonary vein; U: Ureter; V: Vacuole Phylogenetic analysis of Rpob gene sequence reveals that this infectious bacteria clades within Paenibacillus genus.