Britta Bueker

Chris Eberlein

Pierre P Gladieux

Angela Schaefer

Alodie Snirc

Dominic J Bennett

Dominik Begerow

Michael E Hood
email: mhood@amherst.edu

Tatiana Giraud

Distribution and population structure of the anther smut Microbotryum silenes-acaulis parasitizing an arctic-alpine plant

Keywords: admixture, disease prevalence, Microbotryum lychnidis-dioicae, multiple infections, nunatak, selfing

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

INTRODUCTION

The geographic distribution and genetic structure of present-day species result both from dispersal ability and from historical processes such as continental drift and climate changes [START_REF] Hewitt | Some genetic consequences of ice ages, and their role in divergence and speciation[END_REF], as changes in sea level and extensions of ice sheets have affected the availability and connectivity of species´ habitats (Andersen & Borns 1997;[START_REF] Ehlers | The extent and chronology of Cenozoic Global Glaciation[END_REF]. In particular, studies using molecular markers have revealed that the genetic structure of presentday species has been strongly influenced by the oscillations in temperature of the Quaternary [START_REF] Hewitt | Genetic consequences of climatic oscillations in the Quaternary[END_REF].

During glacial periods of the Pleistocene, cold-sensitive species persisted in ice-free "refugia" in southern regions. Long-term isolation of populations in separate refugia fostered population differentiation [START_REF] Bennett | Quaternary refugia of north European trees[END_REF][START_REF] Hewitt | Some genetic consequences of ice ages, and their role in divergence and speciation[END_REF][START_REF] Hewitt | Genetic consequences of climatic oscillations in the Quaternary[END_REF]. Increasing temperatures and decline of ice sheets during interglacial periods enabled range expansions and migration northwards [START_REF] Comes | The effect of Quaternary climatic changes on plant distribution and evolution[END_REF][START_REF] Abbott | History and evolution of the arctic flora: in the footsteps of Eric Hultén[END_REF]. Expansions as part of these recolonization processes were often associated with founder effects, resulting in low levels of genetic diversity in northern regions, while southern regions remained reservoirs of genetic diversity [START_REF] Hewitt | Genetic consequences of climatic oscillations in the Quaternary[END_REF]. In contrast, cold-adapted species were able to survive glacial maxima in more northern areas, at the edge of the ice-sheets, and also in mountains of southern areas (Murray 1995;[START_REF] Abbott | Molecular analysis of plant migration and refugia in the Arctic[END_REF][START_REF] Brochmann | Glacial survival or tabula rasa? The history of North Atlantic biota revisited[END_REF]. Molecular studies have supported the existence of additional, smaller refugia in northern Europe and North America, e.g., in "nunataks", that were mountains completely surrounded by glacial ice (e.g. Stehlik et al. 2002;Westergaard et al. 2011), from which species dispersed when the climate became warmer. The range of arctic-alpine species was thus also fragmented during the ice age, which left footprints Comment citer ce document : Bueker, B., Eberlein, C., Gladieux, P., Schaefer, A., Snirc, A., Bennett, D. J., Begerow, D., Hood, M. E. (Auteur de correspondance), Giraud, T. (2016). Distribution and population structure of the anther smut Microbotryum silenes-acaulis parasitizing an arctic-alpine plant. Molecular Ecology, 25 (3), 811-824. DOI : 10.1111/mec.13512

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as strong genetic population structures [START_REF] Eidesen | Genetic roadmap of the Arctic: plant dispersal highways, traffic barriers and capitals of diversity[END_REF]. Unlike species adapted to temperate climates however, the postglacial recolonization of many cold-adapted species was likely broadfronted, with admixtures between different genetic pools, and therefore with little loss of overall genetic variation [START_REF] Brochmann | The Arctic -an evolutionary freezer?[END_REF].

Recent studies on arctic-alpine species have shown that, beside the impact of post-glacial migration, the genetic structure of present day species varies greatly depending on speciesspecific factors, e.g., dispersal ability and mating system (Taberlet et al. 1998;[START_REF] Abbott | History and evolution of the arctic flora: in the footsteps of Eric Hultén[END_REF]Stewart et al. 2010). In pathogenic organisms, population structure may depend in addition on the distribution of the host (Nieberding et al. 2008). Although a wealth of studies on the genetic diversity of arctic-alpine organisms has been accumulated, they virtually all focus on plant and animal taxa, leaving a striking lack of knowledge in other groups. For instance, only few studies have focused on fungal pathogens or fungi in symbiosis, e.g., mycorrhizae [START_REF] Cripps | Distribution of mycorrhizal types among alpine vascular plant families on the Beartooth Plateau, Rocky Mountains, U.S.A., in reference to large-scale patterns in arctic-alpine habitats[END_REF]); yet such research is important if we are to understand the effects of climate change on disease emergence and spread and broader issues of impact from warming temperatures on biodiversity.

We therefore studied here the population structure of the arctic-alpine distributed pathogen

Microbotryum silenes-acaulis for understanding its population history in relation to climate changes during and after the last glaciation. This specialized basidiomycete fungal pathogen, M.

silenes-acaulis, is found on the plant Silene acaulis [START_REF] Lutz | Anther smuts of Caryophyllaceae: Molecular analyses reveal further new species[END_REF], which occurs in arcticalpine environments in the northern hemisphere [START_REF] Körner | Alpine plant life: functional plant ecology of high mountain ecosystems[END_REF]. Silene acaulis forms large, longlived cushions with hundreds of short stems [START_REF] Jones | Silene acaulis (L.) Jacq[END_REF].

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recolonization from southern European refugia (Spain, Italy, Balkans) after the last glaciation (Vercken et al. 2010). The M. lychnidis-dioicae recolonization history is mostly congruent with that of Silene latifolia (Taylor & Keller 2007), although that pathogen seemed to have persisted in more numerous and smaller refugia than its host (Vercken et al. 2010;[START_REF] Gladieux | Epidemiology and evolution of fungal pathogens in plants and animals[END_REF].

Infections by multiple pathogen genotypes have been reported in M. lychnidis-dioicae and can have important consequences on disease dynamics (Lopez-Villavicencio et al. 2007, 2011;[START_REF] Buono | Influence of multiple infection and relatedness on virulence: Disease dynamics in an experimental plant population and its castrating parasite[END_REF]Susi & Laine 2015).

To investigate the phylogeography of M. silenes-acaulis in relation to its host distribution, we used a combination of herbarium surveys and population genetic analyses using microsatellite markers. Our specific questions were the following: 1) What is the distribution of M. silenesacaulis as compared to its host plant S. acaulis? 2) What is the population structure of M.

silenes-acaulis across its geographical range, and does it allow retracing migration histories in relation to glacial cycles? 3) What is the spatial distribution of the genetic variation at finer scales, including whether infections by multiple pathogen genotypes occur in the same S. acaulis individuals?

MATERIAL AND METHODS

Herbarium surveys for assessing species distribution

To study the distribution of M. silenes-acaulis and its host S. acaulis, a total of 1437 herbarium specimens of S. acaulis were examined. Several previous studies have shown that anther-smut fungi can be detected among herbarium specimens of its hosts; because there is little evidence specimens were recorded.

The distributions of healthy and diseased specimens were compared to the previously published S. acaulis map by [START_REF] Hultén | Atlas of North European vascular plants: North of the tropic of cancer. 3: commentary to the maps: total index[END_REF]. Disease incidence among herbarium specimens was

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STATISTICS 19 (SPSS,Inc,Chicago,Illinois); this analysis included the disease sites and an equal number of randomly selected healthy sites.

Sample collection of M. silenes-acaulis in living populations for population genetic analyses

The analyses of M. silenes-acaulis population structure were performed on a sample of individuals collected from natural, living populations across its range (Fig. 1, Table S1) from 2001 to 2013. Diploid teliospores from host plants within an area of 2 km 2 were considered as a geographic population and spores from a given flower were treated as a single diploid individual (Lopez-Villavicencio et al. 2007).

Genotyping populations of the global dataset

DNAs from all 255 sampled individuals (from populations in North America, Iceland, southern and northern Europe, including Norway and Svalbard), hereafter called the global dataset, were extracted using chelex [START_REF] Giraud | Patterns of within population dispersal and mating of the fungus Microbotryum violaceum parasitising the plant Silene latifolia[END_REF]) and genotyped using 16 microsatellite markers previously available (Table S2) [START_REF] Bucheli | Isolation and characterization of microsatellite loci in the anther smut fungus Microbotryum violaceum[END_REF][START_REF] Giraud | Isolation of 44 polymorphic microsatellite loci in three host races of the phytopathogenic fungus Microbotryum violaceum[END_REF]Giraud et al. 2008).

Only individuals with genotype data for more than 70 % of the markers were retained for analyses, which represented 195 individuals from 39 populations.

Development of microsatellite markers and genotyping populations of the northern-

European dataset

Because of higher sampling density in Norway and Svalbard, we also analyzed these populations separately, and with markers specifically developed from local genotypes, to increase the power of the population structure analyses in these regions.

length was between two and nine bp, which is typically considered as the optimal length range for obtaining variable and scorable markers, 2) they had a minimum length of 20 bp and/or at least four repeats, for increasing the probability that the loci would be polymorphic, and 3) they were perfect repeats of the motif, for decreasing the probability of wrong allele scoring and for maximizing the probability of polymorphism [START_REF] Leese | Analysing intraspecific genetic variation: A practical guide using mitochondrial DNA and microsatellites[END_REF].

Primer binding sites were chosen so that the amplified fragment would range between 100 and 350 bp (Table S3). Primer pairs were designed using PRIMER3 0.4.0 (Rozen & Skaletsky 2000) and prepared by Sigma-Aldrich Life Science (Taufkirchen, Germany). Variation for the designed microsatellite markers was tested by genotyping four specimens from four distinct regions, respectively: Alps, Norway, Svalbard and Alaska. To reduce the occurrence of stutter bands in fragment analyses, the final elongation time in the PCRs was set to 30 minutes [START_REF] Leese | Analysing intraspecific genetic variation: A practical guide using mitochondrial DNA and microsatellites[END_REF]. Markers with multiple amplified fragments were not retained for further genotyping.

After testing different microsatellite combinations using gel electrophoresis, we pooled two markers at most using multiplex PCR, with different fragment lengths and adding the fluorescent

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6-Fluorescein-phosphoramidite (6-FAM™) (Sigma-Aldrich Life Science) to the 5'-end forward primers. Fragment analysis was performed on an ABI3130xl sequencer (Applied Biosystems), using the standard ladder GeneScan-500 ROX™.

After genotyping northern European populations with these markers, we checked for large allele drop out using MICROCHECKER 2.2.3 (Van Ooosterhout et al. 2004) and removed loci displaying extremely large among-population variance using ANIMALFARM 1.0 [START_REF] Landry | Deriving evolutionary relationships among populations using microsatellites and (deltamu)(2): all loci are equal, but some are more equal than others[END_REF]. The program LOSITAN 1.0 [START_REF] Beaumont | Evaluating loci for use in the genetic analysis of population structure[END_REF] was used to detect markers evolving under positive selection, and GENEPOP 4.1 (Rousset 2008) was used to detect markers in linkage disequilibrium, which were then excluded from further analyses. We eventually retained seven of the newly developed markers for the population structure analysis of Norway and Svalbard populations (Table S3). Finally, we retained 110 individuals from 18 populations with no more than 30 % missing data.

Investigation of the occurrence of multiple infections

To investigate the occurrence of multiple infections in S. acaulis, we genotyped spores from three to five different stems per diseased host plant in four of the populations from southern Europe (Table S1). This subset represented 64 flowers from 16 plants, genotyped with the 16 microsatellite markers used for the global dataset.

Genetic diversity

For the global dataset, the degree of genetic variability within the geographic groups North America, southern Europe and northern Europe was assessed by allelic richness (A r ) (Petit et al.

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Population structure

Population genetic structures were investigated with the Bayesian assignment method implemented in STRUCTURE 2. 3 (Pritchard et al. 2000) using the diploid datasets. Ten independent runs were performed for each value of K (number of clusters), with K ranging from one to 20. We used 500,000 iterations after a burn-in period of 100,000. STRUCTURE outputs were processed using STRUCTURE HARVESTER (Earl & von Holdt 2012).

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(Rosenberg 2003). The geographic distributions of genetic clusters were visualized using R package "RgoogleMaps" [START_REF] Loecher | RgoogleMaps and loa: Unleashing R Graphics Power on Map Tiles[END_REF]. Genetic diversity and selfing rates in genetic populations were estimated using GENALEX 6.5 (Peakall & Smouse 2005) and INSTRUCT [START_REF] Gao | A Markov Chain Monte Carlo Approach for joint inference of population structure and inbreeding rates from multilocus genotype data[END_REF], respectively. F ST values among genetic clusters were calculated at K = 3 to K = 8 and visualized with neighbour-joining trees using the R package "ape" (Paradis et al. 2015).

In addition to these model-based methods, we performed a principal component analysis (PCA)

to visualize genetic patterns without any assumption on panmixia (R packages "vegan" (Oksanen et al. 2015), "rgl" (Adler et al. 2015) and "scatterplot3d" (Ligges et al. 2015). For each value of K from two to ten, individuals were assigned to a cluster if their membership coefficient in this cluster was higher than 0.7.

RESULTS

Distribution and demographic data

Herbarium specimens of S. acaulis were examined from throughout the species range and largely conformed to the species map provided by [START_REF] Hultén | Atlas of North European vascular plants: North of the tropic of cancer. 3: commentary to the maps: total index[END_REF] (Fig. 1), i.e., an alpine/circum-polar distribution, including more southern extensions in mountain ranges, such as the Rocky Mountains, and a gap in the northern Eurasia regions of the Sakha Republic.

A total of 1,437 herbarium specimens were examined, including 41 (2.9%) that were found to be affected by the anther-smut disease. Peak collection period of the plant S. acaulis was in the 1930s, consistent with previous studies on other plant species [START_REF] Antonovics | Herbarium studies on the distribution of anther-smut fungus (Microbotryum violaceum) and Silene species (Caryophyllaceae) in the eastern United States[END_REF]. There was no significant difference in the distribution of collection times (years) between diseased and

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healthy samples (Kolmogorov-Smirnov two sample test, p-value = 0.545), indicating no recent change in disease incidence. As previously observed for other Silene herbarium specimens [START_REF] Antonovics | Herbarium studies on the distribution of anther-smut fungus (Microbotryum violaceum) and Silene species (Caryophyllaceae) in the eastern United States[END_REF], there was no indication that collectors had been aware of the disease status of the plants and would have biased collection towards healthy or diseased plants. In particular, no written mention of diseased was recorded on herbarium sheets. Excluding specimens with illegible or ambiguous locality information, 790 distinct collection localities were identified, including 36 with anther smut infection.

Anther-smut disease was found to be widespread within the host's geographic range, with no major region being free of infections. However, the distribution of anther smut disease showed significant patterns among the collection sites in two ways. First, the disease tended to be found more often in regions with low densities of S. acaulis collection sites (Fig. 2); none of the 1,000 randomly sampled sets of healthy sites had average densities of nearby sites, in radii at 50, 100 or 200 km, as low as that observed for the set of diseased sites (p-value < 0.001). Second, the proportion of sites that contained diseased plants increased in more northern regions (Fig. 3).

This pattern of greater disease incidence in the northern latitudes was despite the occurrence of greater numbers of samples per site in more southern localities (1.7 samples/site for each of the two southern latitudinal quartiles, and 1.4 and 1.3 samples/site for the two more northern quartiles, respectively).

While host collection site density had a strong latitudinal component (Fig. 3), the number of nearby collection sites was significantly determined by disease status independently of the effect of latitude, with disease being found in areas of lower host site density. The number of nearby sites within a radius of 100km was indeed predicted from the generalized linear model by

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latitude (Wald X 2 = 113.32, p-value < 0.001) and disease status (Wald X 2 = 5.66, p-value = 0.017), while the interaction term was non-significant (Wald X 2 = 0.43, p-value = 0.514).

Genetic variation in populations and within individual host plants

We detected 99 different alleles overall across the 16 microsatellite markers used on the global dataset, including M. silenes-acaulis samples from 195 different plants across 42 populations.

Typically, three to nine alleles per locus were observed, except for the E18 marker, which displayed 24 alleles (Table S2). For all markers, observed heterozygosity (H O ) showed significantly lower values than the expected heterozygosity (H E ) (p < 0.001); this tendency of heterozygote deficiency was also indicated by positive and high G IS values (Table 1, Table S2).

Expected heterozygosity (H E ) and allelic richness (A r ) were highest in southern European Multiple infections were detected in the 16 plants from populations in the Alps for which spores from multiple stems had been genotyped (mean number of stems analyzed = 4). A mean of 3.25 genotypes were detected per plant, i.e., nearly every stem analyzed bore a specific genotype.

There was no significant correlation between the average number of genotypes per individual

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and genetic diversity (H E and A r ) of a population (H E : r = 0.80, p-value = 0.333; A r : r = 0.63, pvalue = 0.368).

Population structure

We first used the Bayesian methods implemented in the STRUCTURE software to investigate the population structure in the global dataset. There was a constant increase of the likelihood Ln(Pr(X|K)) for the number of clusters (K) ranging from two to 20 clusters; nevertheless, after K = 8 the rate of increase in probability became markedly smaller. In addition, the barplots showed that for K > 8 each new cluster appeared completely admixed and therefore did not correspond to a genuine population structure. Together this indicated that K = 8 was the finest genetic structure we could detect with our dataset.

At K = 3, a clear separation was observed between North America, northern Europe and southern Europe. Populations from North America, Norway and the Pyrenees were mostly assigned to a single cluster each (blue, yellow and green clusters, respectively), while individuals from the Alps were assigned to either one of these three clusters (Figs. 4 and5).

At K = 8 the North American populations were mainly assigned to two clusters (blue and orange) (Figs. 4 and5). The blue cluster was exclusively found in North America, and mostly in Colorado, while the orange cluster occurred mostly in Alaska but also in the Alps. In Norway, two clusters were found (yellow and brown), scattered across all Norwegian regions. Samples from central Alps were mainly assigned to three clusters (red, orange and violet), also with some individuals from the western Alps. Individuals from southern Alps were assigned to two clusters, one being restricted to this area (light green), while the other cluster could be also found in

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substantial membership proportions in northern and southern European populations.

INSTRUCT, which jointly estimates selfing rates and population structure, inferred a similar population structure as the STRUCTURE software. For K = 5, i.e., the highest K-value for which cluster assignment was clear, the lowest selfing rates were found in the dark green cluster (0.37), in the southern Alps, while the highest selfing rate was inferred in the red cluster (0.67), in central Alps. Allelic richness (A r ) was lowest in the Norway yellow cluster (A r = 1.69) and highest in the dark green cluster in the southern Alps (A r = 3.51) (Table 2).

PCA analyses displayed patterns largely consistent with the Bayesian clustering (Fig. S2). PCA and pairwise F ST values (Table 3; Figs. 5 andS2) further revealed a close relationship between the two clusters identified in Norway/Svalbard, which also appeared genetically close to the red cluster in central Alps. The blue cluster, which occurred mostly in the North American populations, appeared genetically the most distant.

Genetic structure of Norway/Svalbard populations

For the populations from Norway and Svalbard, the set of the seven new microsatellite markers showed similar levels of genetic variability of M. silenes-acaulis as the previously developed markers. The average number of alleles per population indeed ranged from 1.6 to 2.14 across the 110 individuals collected in the 18 populations from Norway and Svalbard populations.

Observed heterozygosity (H O ) was lower than expected heterozygosity (H E ) for all seven loci, and the tendency of homozygosity excess was also indicated by high G IS values, with an average of 0.65 (Table 1, Table S2).

STRUCTURE results displayed an increase of the likelihood Ln(Pr(X|K) for increasing K up to K = 9, but the increase became smaller above K = 4. The Evanno-method thus indicated that the

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subdivision in four genetic clusters was the strongest structure (Fig. S1). Results of the structure analysis supported the existence of four genetic clusters, that were however not strongly associated with geographical regions (Figs. S3 and S4). Each cluster was indeed present in all regions, although the yellow cluster was mainly restricted to Svalbard populations (Figs. S3 andS4). This cluster appeared to be the most genetically distant based on F ST values (Table S4; Fig. S4). The selfing rates inferred by INSTRUCT varied from 0.72 to 0.81 for the four genetic clusters (Table 4).

DISCUSSION

The present study combined herbarium surveys and population genetics for understanding the effects of climate and environmental factors on the distribution and population subdivision a plant pathogen species with an arctic-alpine distribution.

Distribution of Silene acaulis and anther-smut disease

Herbarium surveys allowed mapping the distribution of the plant S. acaulis and of its anther smut pathogen M. silenes-acaulis at a broad geographic scale. The geographical range inferred for S.

acaulis was largely in agreement with the herbarium and records-based species map provided by [START_REF] Hultén | Atlas of North European vascular plants: North of the tropic of cancer. 3: commentary to the maps: total index[END_REF]. This supports the utility of herbarium collections as resources for estimating worldwide species ranges, including for fungal pathogens preserved with their hosts [START_REF] Antonovics | Herbarium studies on the distribution of anther-smut fungus (Microbotryum violaceum) and Silene species (Caryophyllaceae) in the eastern United States[END_REF][START_REF] Hood | Distribution of the anther-smut pathogen Microbotryum on species of the Caryophyllaceae[END_REF]. The absence of notes about anther-smut disease on specimens in herbarium surveys further confirmed that collectors do not appear to detect infections on the plants, which has been also shown by other studies (Hood & Antonovics 2003),

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Comment citer ce document : Bueker, B., Eberlein, C., Gladieux, P., Schaefer, A., Snirc, A., Bennett

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suggesting that collection bias has likely been minimal. Thus, herbaria surveys are useful for inferring disease range and rates of disease in nature [START_REF] Antonovics | Herbarium studies on the distribution of anther-smut fungus (Microbotryum violaceum) and Silene species (Caryophyllaceae) in the eastern United States[END_REF].

Anther-smut pathogen was found throughout the geographic range of S. acaulis with an incidence of 2.9%, which is twice the average rate compared to other Silene hosts of anther smuts estimated from broader herbarium surveys [START_REF] Hood | Distribution of the anther-smut pathogen Microbotryum on species of the Caryophyllaceae[END_REF]. Part of the explanation for the high levels of disease on S. acaulis may come from the very long-lived nature of the plants (several hundred years; [START_REF] Forbis | Seedling establishment and life history trade-offs in alpine plants[END_REF] in combination with a systemic infection from which S. acaulis typically rarely recovers [START_REF] Marr | Spatial and temporal pattern of a pollinator-transmitted pathogen in a long-lived perennial, Silene acaulis[END_REF]. Using a model that incorporates dynamics of anther smut transmission, [START_REF] Bruns | Rate of resistance evolution and polymorphism in longand short-lived hosts: Brief communication[END_REF] recently showed that host longevity is associated with higher equilibrium disease prevalence (i.e., proportion of host individuals that are diseased within populations), which would result in the disease being more likely to be sampled when herbarium materials are collected.

In addition, there was significantly greater incidence of anther-smut disease in more northern latitudes and where the host locations were less dense, indicating an influence of environmental factors and/or host demographic structure on the pathogen distribution. While there is a possibility that far northern regions have lower densities of collection sites because of biases in sampling efforts, previous herbarium surveys have suggested the importance of host demography on disease prevalence, where the disease was more common in host species exhibiting higher levels of localized endemicity [START_REF] Hood | Distribution of the anther-smut pathogen Microbotryum on species of the Caryophyllaceae[END_REF] or in more isolated localities [START_REF] Antonovics | Herbarium studies on the distribution of anther-smut fungus (Microbotryum violaceum) and Silene species (Caryophyllaceae) in the eastern United States[END_REF]. A negative effect of host site density on anther smut incidence may appear surprising, as higher host density is usually thought to promote sustainability of pathogen populations [START_REF] Anderson | Population biology of infectious diseases: Part I[END_REF]. However, field studies of Microbotryum on the plant Viscaria alpine also found higher disease prevalence in regions of more rarefied host populations (Carlsson-

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This article is protected by copyright. All rights reserved. [START_REF] Carlsson-Granér | The impact of host longevity on disease transmission: host-pathogen dynamics and the evolution of resistance[END_REF][START_REF] Carlsson-Granér | Patterns of disease and host resistance in spatially structured systems[END_REF]. This was explained by lower population connectedness preventing the maintenance and selection for resistant host genotypes. In S.

acaulis, additional losses of resistant alleles might have occurred due to founder effects during northward recolonization after the last glaciation. Resistance is indeed known to display extensive genetic variation within Silene hosts [START_REF] Biere | Host adaptation in the anther smut fungus Ustilago violacea Version postprint Comment citer ce document[END_REF][START_REF] Cafuir | Tissue culture and quantification of individual level resistance to anther smut disease in Silene vulgaris[END_REF]) while infection ability varies little within Microbotryum species [START_REF] Kaltz | Local maladaptation in the anther-smut Version postprint Comment citer ce document[END_REF]; it would therefore be expected that founder effects in northern areas would have less of an impact on infection ability in the pathogen than on resistance within the host. In addition to these possible influences on resistance polymorphisms, climatic effects of latitude may play a direct role in determining anther smut distributions, through a sensitivity of the pathogen to warmer conditions.

Microbotryum as a genus tends to exhibit an association between high frequencies of disease and mountainous regions [START_REF] Hood | Distribution of the anther-smut pathogen Microbotryum on species of the Caryophyllaceae[END_REF], and it has been assumed that, like many fungal diseases, it is favored by cooler temperatures. The recent study by [START_REF] Abbate | Elevational disease distribution in a natural plant-pathogen system: insights from changes across host populations and climate[END_REF] actually demonstrated that Microbotryum species affecting S. vulgaris were more likely to be found in host populations where overall temperatures were lower.

Population structure of M. silenes-acaulis in comparison to other non-arctic-alpine species

The genetic diversity of M. silenes-acaulis was low overall compared to other Microbotryum species, such as M. lychnidis-dioicae and M. silenes-dioicae (Vercken et al. 2010). This could be due to M. silenes-acaulis having a relatively small effective population size, due to a high degree

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habitats that may facilitate the exchange and maintenance of allelic diversity in their anther smuts.

Additional host-specific features of S. acaulis may also be expected to affect pathogen genetic diversity within host populations and even within individual host plants through multiple infections, as is known to occur in M. lychnidis-dioicae [START_REF] Hood | Dynamics of Multiple Infection and Within Host Competition by the Anther Smut Pathogen[END_REF]Lopez-Villavicencio et al. 2007). In particular, this study shows that for some samples from the Alps most stems within a given plant carry different M. silenes-acaulis genotypes. The long lifespan and the high number of stems of the host plant in particular are expected to favor the occurrence of multiple infections, as it increases the probability that different genotypes are deposited on a given host individual plant and that different genotypes occupy different stems. [START_REF] Marr | Spatial and temporal pattern of a pollinator-transmitted pathogen in a long-lived perennial, Silene acaulis[END_REF] estimated that their particular S. acaulis population under-study could have persisted for approximately 2000 years based on past geological data, and S. acaulis cushions can bear a multitude of perennial stems [START_REF] Jones | Silene acaulis (L.) Jacq[END_REF].

Our results suggested a predominantly selfing mating system in M. silenes-acaulis. Although selfing is typical of Microbotryum species (Giraud et al. 2008), estimated selfing rates were not as severe as previously observed in M. lychnidis-dioicae and M. silenes-dioicae, for example (Vercken et al. 2010;[START_REF] Gladieux | Epidemiology and evolution of fungal pathogens in plants and animals[END_REF]. Opportunities to encounter a mating partner for outcrossing may be higher in M. silenes-acaulis than in other Microbotryum species due to the long life span, high number of stems per plant, persistence of host populations, and low disease recovery rates that characterize S. acaulis [START_REF] Marr | Spatial and temporal pattern of a pollinator-transmitted pathogen in a long-lived perennial, Silene acaulis[END_REF]. Alternatively, arctic alpine environment and long generation time [START_REF] Bruns | Rate of resistance evolution and polymorphism in longand short-lived hosts: Brief communication[END_REF]) may affect mating system and outcrossing rates in fungal systems in a similar way as described for plants. In plants, artic-alpine environments have been suggested to increase selfing rates due to inadequate pollinator services

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in harsh environments, stress-induced increase in levels of self-compatibility and a paucity of self-incompatibility alleles within smaller populations [START_REF] Karron | New perspectives on the evolution of plant mating systems[END_REF][START_REF] Levin | Mating system shifts on the trailing edge[END_REF].

Genetic subdivision and migration in M. silenes-acaulis

Long-standing physical barriers (e.g. oceans or mountains) are known to limit dispersal and induce differentiation of populations [START_REF] Abbott | History and evolution of the arctic flora: in the footsteps of Eric Hultén[END_REF][START_REF] Eidesen | Genetic roadmap of the Arctic: plant dispersal highways, traffic barriers and capitals of diversity[END_REF]. Our results revealed strong differentiation in M. silenes-acaulis between populations from North America and Europe, as previously reported in other arctic-alpine distributed species [START_REF] Abbott | History and evolution of the arctic flora: in the footsteps of Eric Hultén[END_REF][START_REF] Alsos | Frequent long-distance plant colonization in the changing Arctic[END_REF]Westergaard et al. 2011). Genetic footprints of long-distance dispersal were nevertheless detected, as North American individuals assigned to European clusters. This feature is shared with several arctic-alpine plant species, for which genetic data have provided ample evidence for extreme colonization abilities, in many cases despite the lack of obvious adaptations to long-distance dispersal [START_REF] Brochmann | The Arctic -an evolutionary freezer?[END_REF]. It remains speculation whether this is human-mediated by importing plants as ornamentals (e.g., Farr et al.

1989) or if long-distance dispersal of seeds of S. acaulis occurs more regular e.g. by seed transfer in feathers of birds (e.g., [START_REF] Lira-Noriega | The roles of history and ecology in chloroplast phylogeographic patterns of the bird-dispersed plant parasite Phoradendron californicum (Viscaceae) in the Sonoran Desert[END_REF].

The biological features of Microbotryum concerning long-distance dispersal similarly raise questions, because they are obligate, specialized pathogens that are neither transmitted by the seeds nor persistent as contaminants of the environment [START_REF] Begerow | The Mycota. 7. Part A. Systematics and evolution[END_REF]. However, the case of the temperate species M. lychnidis-dioicae -that was introduced in North Americashows that even a vector-borne pathogen can successfully invade a new continent multiple times independently [START_REF] Fontaine | History of the invasion of the anther smut pathogen on Silene latifolia in North America[END_REF][START_REF] Gladieux | The population biology of fungal invasions[END_REF].

Moreover the present study revealed a clear genetic differentiation between southern and

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northern European populations, similar to the population structure of its host S. acaulis (Mikhailova et al. 2010). During the last glaciation, conditions in central Europe were not suitable even for cold-adapted organisms, so that populations have remained separated for long in distinct refugia, which has fostered population differentiation. Multiple previous studies have similarly revealed footprints of distinct glacial refugia in Europe [START_REF] Hewitt | Some genetic consequences of ice ages, and their role in divergence and speciation[END_REF][START_REF] Hewitt | Genetic consequences of climatic oscillations in the Quaternary[END_REF], including in artic-alpine plants [START_REF] Abbott | History and evolution of the arctic flora: in the footsteps of Eric Hultén[END_REF][START_REF] Eidesen | Genetic roadmap of the Arctic: plant dispersal highways, traffic barriers and capitals of diversity[END_REF]. Only few studies have investigated population genetic structures at large scales in natural plant pathogens (i.e., noncrop disease), and some of them have found subdivision between continents, e.g., in Melampsora larici-populina [START_REF] Barrès | Genetic structure of the poplar rust fungus Melampsora larici-populina: Evidence for isolation by distance in Europe and recent founder effects overseas[END_REF].

The differentiation in southern Europe of several distinct genepools in separated refugia also explains the highest levels of genetic variation in M. silenes-acaulis in southern Europe. Postglacial migration from several peripheral refugia or even "nunataks" would then have resulted in the observed co-occurrence of several clusters within small areas as well as admixture (Schönswetter et al. 2006). High genetic diversity and cluster richness in southern Europe are features shared with several other arctic-alpine species i.e. Arabis alpina [START_REF] Koch | Three times out of Asia Minor: the phylogeography of Arabis alpina L. (Brassicaceae): Phylogeography of Arabis alpina[END_REF] and

Ranunculus glacialis (Schönswetter et al. 2003).

The lower genetic diversity and the occurrence of distinct clusters in the more northern populations of M. silenes-acaulis in Europe would result from demographic bottlenecks following recolonization from more southern refugia. This is supported by the northern clusters being derived from clusters occurring in southern Europe in the NJ trees. Similar patterns have been observed for the host species S. acaulis [START_REF] Abbott | Molecular diversity and derivations of populations of Silene acaulis and Saxifraga oppositifolia from the high Arctic and more southerly latitudes[END_REF][START_REF] Mikhaylova | ) Jacq. (Caryophyllaceae) in Version postprint Comment citer ce document[END_REF]) and other organisms [START_REF] Abbott | History and evolution of the arctic flora: in the footsteps of Eric Hultén[END_REF].

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CONCLUSION

This study combined surveys of natural history collections and population genetics to infer the global biogeography of a fungal pathogen with arctic-alpine distribution. The pathogen appears to have a nearly ubiquitous presence in regions occupied by the hosts S. acaulis. The significantly greater incidence of the pathogen in more northern and more rarefied host locations reveals a major within-species variation in disease risk that has been rarely observed in nonagricultural or domesticated systems. This may be due to lower genetic variation for resistance in northern regions due to post-glacial recolonization and a paucity of host population connectedness. We additionally found evidence for strong population structure within M. silenesacaulis, suggesting persistence in distinct glacial refugia, followed by recolonization from multiple differentiated populations and recent migration events. These overall patterns are consistent with those reported in the host S. acaulis [START_REF] Abbott | Molecular diversity and derivations of populations of Silene acaulis and Saxifraga oppositifolia from the high Arctic and more southerly latitudes[END_REF][START_REF] Mikhaylova | ) Jacq. (Caryophyllaceae) in Version postprint Comment citer ce document[END_REF]. This supports the very close and long-term association of the obligate and specialized pathogen on a very long-lived herbaceous host throughout integrated processes of recolonization after the last glaciation.

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  A well-studied relative of M. silenes-acaulis, Microbotryum lychnidis-dioicae, causes anther smut on the temperate host Silene latifolia, and comparable population genetics studies indicate a clear pattern of Comment citer ce document : Bueker, B., Eberlein, C., Gladieux, P., Schaefer, A., Snirc, A., Bennett, D. J., Begerow, D., Hood, M. E. (Auteur de correspondance), Giraud, T. (2016). Distribution and population structure of the anther smut Microbotryum silenes-acaulis parasitizing an arctic-alpine plant. Molecular Ecology, 25 (3), 811-824. DOI : 10.1111/mec.13512

  analysed in the context of collection site latitude and density of nearby collection sites. The effects of latitude were analysed by separating the samples into equal sized groups based on quartiles from southern to northern latitudes; quartile latitude boundaries of <44, 49, 62, and the maximum observed site of 82. For defining local densities, latitude and longitude information were translated into pairwise linear distances between sites. The number of collection sites near diseased sites (within radii of 50, 100, or 200 km) was compared to an equal number (N = 36) of randomly selected healthy collection sites. One thousand randomly assembled sets of 36 healthy collection sites were used to assess statistical significance of differences between average densities of collection sites near to diseased and healthy localities. Latitude and disease status, and their interaction term, were analysed for the response variable of density of host collection sites using the generalized linear model approach with Poisson log link function in SPSS Version postprint Comment citer ce document : Bueker, B., Eberlein, C., Gladieux, P., Schaefer, A., Snirc, A., Bennett, D. J., Begerow, D., Hood, M. E. (Auteur de correspondance), Giraud, T. (2016). Distribution and population structure of the anther smut Microbotryum silenes-acaulis parasitizing an arctic-alpine plant. Molecular Ecology, 25 (3), 811-824. DOI : 10.1111/mec.13512

  markers 5-Carboxytetramethyl-rhodamine (TAMRA™), 5-Tetrachloro-Fluorescein (TET™) and Comment citer ce document : Bueker, B., Eberlein, C., Gladieux, P., Schaefer, A., Snirc, A., Bennett, D. J., Begerow, D., Hood, M. E. (Auteur de correspondance), Giraud, T. (2016). Distribution and population structure of the anther smut Microbotryum silenes-acaulis parasitizing an arctic-alpine plant. Molecular Ecology, 25 (3), 811-824. DOI : 10.1111/mec.13512

  Comment citer ce document : Bueker, B., Eberlein, C., Gladieux, P., Schaefer, A., Snirc, A., Bennett, D. J., Begerow, D., Hood, M. E. (Auteur de correspondance), Giraud, T. (2016). Distribution and population structure of the anther smut Microbotryum silenes-acaulis parasitizing an arctic-alpine plant. Molecular Ecology, 25 (3), 811-824. DOI : 10.1111/mec.13512

  1998), unbiased expected heterozygosity (H E ), observed heterozygosity (H O ), and Nei´s standardized fixation index (G´S T )(Nei 1987;[START_REF] Meirmans | Assessing population structure: FST and related measures: Invited technical review[END_REF]. The R package "hierfstat"[START_REF] Goudet | hierfstat: Estimation and tests of hierarchical F-statistics[END_REF]) was used to estimate A r , and GENALEX 6.5(Peakall & Smouse 2005) to estimate H E , H O , and G´S T . The significance of the heterozygosity deficit compared to the expected heterozygosity under Hardy-Weinberg assumptions was tested in GENEPOP 4.2 (Rousset 2008) and G IS coefficient was estimated with the correction of Nei and Chesser (1983) using GENALEX 6.5(Peakall & Smouse 2005). Differences in genetic diversity (H E , A r ) between geographic groups were tested using paired Wilcoxon signed rank tests. The relationship between the level of multiple infections and population genetic diversity was assessed by testing the significance of the correlation between the number of genotypes detected per plant and population diversity (H E , A r ) using the Spearman´s rank coefficient. All statistical tests were performed using RSTUDIO 3.0.2 (RStudio Team 2015).

  CLUMPP 1.1.2 (Jackobsson & Rosenberg 2007) was used to align independent runs with the same clustering mode, with random input order, 10,000 permutations, and the Greedy algorithm for K ≤ 9 and the Large-Greedy algorithm for K > 9. Barplots with cluster-membership coefficients for all individuals and geographic populations were generated with the program DISTRUCT 1.1 Comment citer ce document : Bueker, B., Eberlein, C., Gladieux, P., Schaefer, A., Snirc, A., Bennett, D. J., Begerow, D., Hood, M. E. (Auteur de correspondance), Giraud, T. (2016). Distribution and population structure of the anther smut Microbotryum silenes-acaulis parasitizing an arctic-alpine plant. Molecular Ecology, 25 (3), 811-824. DOI : 10.1111/mec.13512

  Comment citer ce document : Bueker, B., Eberlein, C., Gladieux, P., Schaefer, A., Snirc, A., Bennett, D. J., Begerow, D., Hood, M. E. (Auteur de correspondance), Giraud, T. (2016). Distribution and population structure of the anther smut Microbotryum silenes-acaulis parasitizing an arctic-alpine plant. Molecular Ecology, 25 (3), 811-824. DOI : 10.1111/mec.13512

  document : Bueker, B., Eberlein, C., Gladieux, P., Schaefer, A., Snirc, A., Bennett, D. J., Begerow, D., Hood, M. E. (Auteur de correspondance), Giraud, T. (2016). Distribution and population structure of the anther smut Microbotryum silenes-acaulis parasitizing an arctic-alpine plant. Molecular Ecology, 25 (3), 811-824. DOI : 10.1111/mec.13512

  populations (H E = 0.45, A r = 4.89), lowest in northern European populations (H E = 0.18, A r = 2.65) and intermediate in North America (H E = 0.35, A r = 3.76) (Table1). Paired Wilcoxon signed rank tests indicated significant differences between southern Europe and northern Europe (H E : W = 4, p-value < 0.001, A r : W = 136, p-value = 0.031) and between North America and northern Europe (H E : W = 4, p-value = 0.009; A r : W = 129, p-value < 0.001), while the values of southern Europe and North America were significantly different only for A r (H E : W = 81, pvalue = 0.244, A r : W = 118, p-value = 0.008).

  Comment citer ce document : Bueker, B., Eberlein, C., Gladieux, P., Schaefer, A., Snirc, A., Bennett, D. J., Begerow, D., Hood, M. E. (Auteur de correspondance), Giraud, T. (2016). Distribution and population structure of the anther smut Microbotryum silenes-acaulis parasitizing an arctic-alpine plant. Molecular Ecology, 25 (3), 811-824. DOI : 10.1111/mec.13512

  Iceland and Pyrenees (dark green). Pyrenees and Iceland populations were highly admixed, with Comment citer ce document : Bueker, B., Eberlein, C., Gladieux, P., Schaefer, A., Snirc, A., Bennett, D. J., Begerow, D., Hood, M. E. (Auteur de correspondance), Giraud, T. (2016). Distribution and population structure of the anther smut Microbotryum silenes-acaulis parasitizing an arctic-alpine plant. Molecular Ecology, 25 (3), 811-824. DOI : 10.1111/mec.13512

  Comment citer ce document : Bueker, B., Eberlein, C., Gladieux, P., Schaefer, A., Snirc, A., Bennett, D. J., Begerow, D., Hood, M. E. (Auteur de correspondance), Giraud, T. (2016). Distribution and population structure of the anther smut Microbotryum silenes-acaulis parasitizing an arctic-alpine plant. Molecular Ecology, 25 (3), 811-824. DOI : 10.1111/mec.13512

  , D. J., Begerow, D., Hood, M. E. (Auteur de correspondance), Giraud, T. (2016). Distribution and population structure of the anther smut Microbotryum silenes-acaulis parasitizing an arctic-alpine plant. Molecular Ecology, 25 (3), 811-824. DOI : 10.1111/mec.13512

  ., Eberlein, C., Gladieux, P., Schaefer, A., Snirc, A., Bennett, D. J., Begerow, D., Hood, M. E. (Auteur de correspondance), Giraud, T. (2016). Distribution and population structure of the anther smut Microbotryum silenes-acaulis parasitizing an arctic-alpine plant. Molecular Ecology, 25 (3), 811-824. DOI : 10.1111/mec.13512

  of endemicity for arctic and alpine environments. In comparison, population size and connectedness in the widespread Silene hosts S. latifolia and S. dioica have much more generalist Comment citer ce document : Bueker, B., Eberlein, C., Gladieux, P., Schaefer, A., Snirc, A., Bennett, D. J., Begerow, D., Hood, M. E. (Auteur de correspondance), Giraud, T. (2016). Distribution and population structure of the anther smut Microbotryum silenes-acaulis parasitizing an arctic-alpine plant. Molecular Ecology, 25 (3), 811-824. DOI : 10.1111/mec.13512

  Comment citer ce document : Bueker, B., Eberlein, C., Gladieux, P., Schaefer, A., Snirc, A., Bennett, D. J., Begerow, D., Hood, M. E. (Auteur de correspondance), Giraud, T. (2016). Distribution and population structure of the anther smut Microbotryum silenes-acaulis parasitizing an arctic-alpine plant. Molecular Ecology, 25 (3), 811-824. DOI : 10.1111/mec.13512

  Comment citer ce document : Bueker, B., Eberlein, C., Gladieux, P., Schaefer, A., Snirc, A., Bennett, D. J., Begerow, D., Hood, M. E. (Auteur de correspondance), Giraud, T. (2016). Distribution and population structure of the anther smut Microbotryum silenes-acaulis parasitizing an arctic-alpine plant. Molecular Ecology, 25 (3), 811-824. DOI : 10.1111/mec.13512

  ., Eberlein, C., Gladieux, P., Schaefer, A., Snirc, A., Bennett, D. J., Begerow, D., Hood, M. E. (Auteur de correspondance), Giraud, T. (2016). Distribution and population structure of the anther smut Microbotryum silenes-acaulis parasitizing an arctic-alpine plant. Molecular Ecology, 25 (3), 811-824. DOI : 10.1111/mec.13512

Figure 1 .

 1 Figure1. Distribution map of healthy (green points) and diseased (red points) Silene acaulis specimen observed in herbaria. The distribution map of S. acaulis after Hulten Fries (1986) is shown in green. Blue points represent regions from where populations for present study were sampled.

Figure 2 .

 2 Figure 2. Density of Silene acaulis localities near diseased and healthy herbarium collection sites. Sites are sorted by the number of other collections sites within a 100 km radius around diseased sites (red line) and around randomly selected healthy sites (grey lines; first 40 random healthy sites shown).

Figure 3 .

 3 Figure 3. Demographic and disease incidence variation in Silene acaulis across southern to northern latitudes. Herbarium localities (N = 790) were divided into latitude quartiles for analysis and density of herbarium collection sites was based upon 100km radii.

Figure 4 .

 4 Figure 4. Genetic clustering of Microbotryum silenes-acaulis genotypes for the worldwide data set (N = 195) under the assumptions from K = 3 to K = 8 clusters. Each vertical bar represents an individual and the colors indicate the proportion of its genotype assigned to the different clusters.

Figure 5 .

 5 Figure 5. Maps showing the mean assignment proportions per locality estimated from the STRUCTURE analyses at K = 3 (a) and K = 8 (b). The square at the bottom is a zoom on the three localities in the Alps. Neighbour-joining trees illustrate the genetic distances among clusters based on FST distances.

  ., Eberlein, C., Gladieux, P., Schaefer, A., Snirc, A., Bennett, D. J., Begerow, D., Hood, M. E. (Auteur de correspondance), Giraud, T. (2016). Distribution and population structure of the anther smut Microbotryum silenes-acaulis parasitizing an arctic-alpine plant. Molecular Ecology, 25 (3), 811-824. DOI : 10.1111/mec.13512

  ., Eberlein, C., Gladieux, P., Schaefer, A., Snirc, A., Bennett, D. J., Begerow, D., Hood, M. E. (Auteur de correspondance), Giraud, T. (2016). Distribution and population structure of the anther smut Microbotryum silenes-acaulis parasitizing an arctic-alpine plant. Molecular Ecology, 25 (3), 811-824. DOI : 10.1111/mec.13512

Table 1 .

 1 MEH analysed herbarium specimens. MEH and TG performed the sampling in the global dataset. CE developed the second set of markers and performed the samling in Norway and Svalbard. ASch helped with the design of the second marker set and designed the sampling strategy in Norway and Svalbard. DB coordinated the sampling and analysis of the second set of markers. DJB and AS genotyped the global dataset. PG and DJB carried out preliminary analyses of a first version of the dataset. BB performed the final analyses of population genetics for both datasets. PG supervised population genetics analyses. BB, MEH, PG and TG wrote the manuscript, with contributions by all authors. TG, MEH and DB supervised the whole project. Polymorphism in Microbotryum silenes-acaulis from different geographic regions.

	AUTHOR CONTRIBUTIONS

Bueker, B., Eberlein, C., Gladieux, P., Schaefer, A., Snirc, A., Bennett, D. J., Begerow, D., Hood, M. E. (Auteur de correspondance), Giraud, T. (2016). Distribution and population structure of the anther smut Microbotryum silenes-acaulis parasitizing an arctic-alpine plant. Molecular Ecology, 25 (3), 811-824. DOI : 10.1111/mec.13512

Accepted Article

This article is protected by copyright. All rights reserved. 1 : using sixteen microsatellite markers developed in

Giraud et al. 2008 and Bucheli et al. 1998, 2

: using the seven microsatellite markers developed in this study. + Average across markers. ** p-value < 0.05; ** p-value < 0.01.

Comment citer ce document : Bueker, B., Eberlein, C., Gladieux, P., Schaefer, A., Snirc, A., Bennett, D. J., Begerow, D., Hood, M. E. (Auteur de correspondance),

Giraud, T. (2016)

. Distribution and population structure of the anther smut Microbotryum silenes-acaulis parasitizing an arctic-alpine plant. Molecular Ecology, 25 (3), 811-824. DOI : 10.1111/mec.13512

Table 2 .

 2 Number of Microbotryum silenes-acaulis individuals, diversity (allelic richness A r and expected heterozygosity H E ) and selfing rates for the STRUCTURE clusters with K = 5 in the whole dataset of Microbotryum silenes-acaulis.

	N	A r	H E	Selfing rate

+ Average across the sixteen microsatellite markers developed in

Giraud et al. 2008 and[START_REF] Bucheli | Isolation and characterization of microsatellite loci in the anther smut fungus Microbotryum violaceum[END_REF]

; N: Number of individuals, A r : Allelic richness; H E : Expected heterozygosity

Table 3 .

 3 Genetic differentiation (F ST ) values between Microbotryum silenes-acaulis genetic clusters at K = 5.

		Dark
	Blue Yellow	Green Red Orange

Comment citer ce document : Bueker, B., Eberlein, C., Gladieux, P., Schaefer, A., Snirc, A., Bennett, D. J., Begerow, D., Hood, M. E. (Auteur de correspondance), Giraud, T. (2016). Distribution and population structure of the anther smut Microbotryum silenes-acaulis parasitizing an arctic-alpine plant. Molecular Ecology, 25 (3), 811-824. DOI : 10.1111/mec.13512

Table 4 .

 4 Number of individuals, diversity (allelic richness A r and expected heterozygosity H E ) and selfing rates for the genetic clusters at K=4 in the Norway/Svalbard populations of Microbotryum silenes-acaulis.

		N	A r	H E	Selfing rate
	Red cluster	28	2.02	0.22	0.81
	Blue cluster	22	2.04	0.18	0.72
	Yellow cluster	23	1.77	0.16	0.74
	Green cluster	25			0.77
			2.12	0.19

+ Average across seven microsatellite markers developed in the present study; N: Number of individuals analyzed, Number of different alleles per locus; A r : Allelic richness; H E : Expected heterozygosity.

ACKNOWLEDGMENTS

TG acknowledges the grant ANR-13-BSV7-0008. MEH acknowledges the National Science Foundation award DEB-1115765. We thank the INRA Research Unit Génétique, Diversité et Ecophysiologie des Céréales (UMR1095), Gentyane platform (Clermont-Ferrand, France) for part of the microsatellite genotyping. This research was also supported by the German Science Foundation (SCHA 1723/1-1) and a grant from the DAAD program "Promos" (German Version postprint Comment citer ce document : Bueker, B., Eberlein, C., Gladieux, P., Schaefer, A., Snirc, A., Bennett, D. J., Begerow, D., Hood, M. E. (Auteur de correspondance), Giraud, T. (2016). Distribution and population structure of the anther smut Microbotryum silenes-acaulis parasitizing an arctic-alpine plant. Molecular Ecology, 25 (3), 811-824. DOI : 10.1111/mec.13512

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Accepted Article

This article is protected by copyright. All rights reserved. Academic Exchange Service). We thank Erin Chung, Janis Antonovics and Mary Gibby for assistance with herbarium surveys and Oliver Röhl, Sascha Lotze-Engelhard and Bill Morris, for collecting and providing samples.

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Adnane Boualem

Afef Lemhemdi

Marie-Agnes Sari

Sarah Pignoly

Christelle Troadec

Fadi Abou Choucha

Ilknur Solmaz

Nebahat Sari

Catherine Dogimont

Abdelhafid Bendahmane

The Andromonoecious Sex Determination Gene Predates the Separation of Cucumis and Citrullus Genera

Understanding the evolution of sex determination in plants requires the cloning and the characterization of sex determination genes. Monoecy is characterized by the presence of both male and female flowers on the same plant. Andromonoecy is characterized by plants carrying both male and bisexual flowers. In watermelon, the transition between these two sexual forms is controlled by the identity of the alleles at the A locus. We previously showed, in two Cucumis species, melon and cucumber, that the transition from monoecy to andromonoecy results from mutations in 1-aminocyclopropane-1-carboxylic acid synthase (ACS) gene, ACS-7/ACS2. To test whether the ACS-7/ACS2 function is conserved in cucurbits, we cloned and characterized ClACS7 in watermelon. We demonstrated co-segregation of ClACS7, the homolog of CmACS-7/CsACS2, with the A locus. Sequence analysis of ClACS7 in watermelon accessions identified three ClACS7 isoforms, two in andromonoecious and one in monoecious lines. To determine whether the andromonoecious phenotype is due to a loss of ACS enzymatic activity, we expressed and assayed the activity of the three protein isoforms. Like in melon and cucumber, the isoforms from the andromonoecious lines showed reduced to no enzymatic activity and the isoform from the monoecious line was active. Consistent with this, the mutations leading andromonoecy were clustered in the active site of the enzyme. Based on this, we concluded that active ClACS7 enzyme leads to the development of female flowers in monoecious lines, whereas a reduction of enzymatic activity yields hermaphrodite flowers. ClACS7, like CmACS-7/CsACS2 in melon and cucumber, is highly expressed in carpel primordia of buds determined to develop carpels and not in male flowers. Based on this finding and previous investigations, we concluded that the monoecy gene, ACS7, likely predated the separation of the Cucumis and Citrullus genera.

Introduction

The majority of angiosperms are hermaphroditic producing exclusively bisexual flowers. Sex determination is a developmental evolutionary process that leads to unisexual flowers in 10% of the species [START_REF] Renner | The relative and absolute frequencies of angiosperm sexual systems: dioecy, monoecy, gynodioecy, and an updated online database[END_REF]. Monoecious species (5-6%) exhibit male and female flowers on the same plant. Dioecious species (5-6%) have separate male and female individuals. There has been concerted effort in the last few decades to pinpoint the master genes controlling sex determination and to understand the evolutionary forces that drive transitions between sexual morphs [START_REF] Diggle | Multiple developmental processes underlie sex differentiation in angiosperms[END_REF][START_REF] Ainsworth | Sex determination in plants[END_REF]. In these investigations, several plant families have emerged as model systems [START_REF] Kubicki | Investigations on sex determination in cucumber (Cucumis sativus L.). V. Genes controlling intensity of femaleness[END_REF][START_REF] Kubicki | Investigations on sex determination in cucumber (Cucumis sativus L.). VII. Andromonoecism and hermaphroditism[END_REF][START_REF] Poole | Inheritance of new sex forms in Cucumis melo L[END_REF][START_REF] Malepszy | Sex determination in cucumber (Cucumis sativus) as a model system for molecular biology[END_REF]. The choice of the Cucurbitaceae is justified by the wide spread of sex among the species of this plant family. Out of 800 investigated species, 460 are monoecious and 340 are dioecious. Throughout evolution of Cucurbitaceae, there have been also numerous shifts between monoecy and other sexual morphs, which makes this plant family a practical model to investigate the molecular mechanisms controlling sex determination.

In monoecious Cucurbitaceae species, such as Cucumis melo, floral primordia are initially bisexual with sex determination occurring by the selective developmental arrest of either the stamen or the carpel, resulting in unisexual flowers [START_REF] Bai | Developmental analyses reveal early arrests of the spore-bearing parts of reproductive organs in unisexual flowers of cucumber (Cucumis sativus L.)[END_REF]. Cloning and characterization of natural mutations leading to transition from monoecy to different sexual morphs in melon and cucumber has brought new insight to the monoecy sex determination pathway. Three master sex genes, Monoecious, Androecious and Gynoecious genes, interact to explain the gender and the position of the flower on the plant [START_REF] Boualem | A conserved mutation in an ethylene biosynthesis enzyme leads to andromonoecy in melons[END_REF][START_REF] Boualem | A conserved ethylene biosynthesis enzyme leads to andromonoecy in two cucumis species[END_REF][START_REF] Boualem | A cucurbit androecy gene reveals how unisexual flowers develop and dioecy emerges[END_REF][START_REF] Martin | A transposon-induced epigenetic change leads to sex determination in melon[END_REF]. In this model the expression of the carpel inhibitor, CmWIP1, encoded by the gynoecious gene, is dependent on non-expression of CmACS11, encoded by the androecious gene. The expression of the stamina inhibitor, CmACS-7, encoded by the monoecious gene is dependent on non-expression of CmWIP1. In monoecious and andromonoecious plants, male flowers develop because of non-expression of CmACS11 that permits the expression of CmWIP1. Although sex determination occurred many times independently during evolution [START_REF] Renner | The relative and absolute frequencies of angiosperm sexual systems: dioecy, monoecy, gynodioecy, and an updated online database[END_REF][START_REF] Barrett | The evolution of plant sexual diversity[END_REF][START_REF] Lewis | The evolution of sex in flowering plants[END_REF], one would expect that related species use common sexual mechanisms. We previously showed that in melon and cucumber, two species that diverged more than 10 million years ago (Mya) share the same pathway leading to monoecy [START_REF] Boualem | A conserved ethylene biosynthesis enzyme leads to andromonoecy in two cucumis species[END_REF]. For instance the melon and the cucumber monoecious gene are orthologs and encode for the rate-limiting enzyme in ethylene biosynthesis, the 1-aminocyclopropane-1-carboxylic acid synthase (ACS). In both species, the ACS genes, referred to as CmACS-7 in melon and CsACS2 in cucumber, are expressed in carpel primordia, and loss of enzymatic activity leads to stamen development [START_REF] Boualem | A conserved ethylene biosynthesis enzyme leads to andromonoecy in two cucumis species[END_REF].

Similarly, the melon and the cucumber androecious genes that encode for an ACS are also orthologs [START_REF] Boualem | A cucurbit androecy gene reveals how unisexual flowers develop and dioecy emerges[END_REF]. In both species, the ACS genes, referred to as CmACS11 in melon and CsACS11 in cucumber, are expressed in carpel primordia, and loss of enzymatic activity leads to stamen development [START_REF] Boualem | A cucurbit androecy gene reveals how unisexual flowers develop and dioecy emerges[END_REF].

In melon and cucumber, two species of the Cucumis genus, our previous studies [START_REF] Boualem | A conserved mutation in an ethylene biosynthesis enzyme leads to andromonoecy in melons[END_REF][START_REF] Boualem | A conserved ethylene biosynthesis enzyme leads to andromonoecy in two cucumis species[END_REF] have shown that the sexual transition from monoecy to andromonoecy results from loss-of-function mutations in CmACS-7 and CsACS2, respectively. In watermelon, a monoecious cucurbit, the recessive a locus controls the andromonoecious sexual phenotype and acts as CmACS-7 and CsACS2 [START_REF] Rosa | The inheritance of flowers types in Cucumis and Citrullus[END_REF]. Recently, a QTL analysis has shown that the major QTL controlling the watermelon andromonoecy is located on chromosome 3 close to the Cla011230 gene encoding an ACC synthase enzyme [START_REF] Prothro | Quantitative trait loci associated with sex expression in an inter-subspecific watermelon population[END_REF].

In the present study, we investigated whether components of the monoecy pathway are conserved between the Cucumis and Citrullus genus that diverged 20 Mya [START_REF] Schaefer | Gourds afloat: a dated phylogeny reveals an Asian origin of the gourd family (Cucurbitaceae) and numerous oversea dispersal events[END_REF]. To test this hypothesis, we cloned the andromonoecious (a) gene from Citrullus lanatus (watermelon) and characterized natural mutations leading to sexual transition between monoecy and andromonoecy. We showed that, like in melon and cucumber, an ACS gene, ClACS7, inhibits stamina development in female flowers and loss of enzymatic activity leads to stamen development. ClACS7 is syntenic to CmACS-7 in melon and CsACS2 in cucumber. Based on this finding, we concluded that the ACS7 function is ancestral to the divergence of the Cucumis and the Citrullus genus.

Materials and Methods

Plant material and segregating population

The genetic linkage analysis of the A locus and ClACS7 was carried out using a segregating population obtained from a cross between a monoecious accession, Citrullus lanatus var. citroides, PI 296341, and an andromonoecious Citrullus lanatus var. lanatus variety Halep Karasi. The resultant F1 was backcrossed with Halep Karasi.

Plants were grown in the greenhouse in summer at Montfavet, France, under standard agronomic conditions and evaluated for the sex of their flowers at each node along the main stem and side branches.

Plant genotyping

To identify plants carrying recombination events, plant DNA was extracted from each individual of the backcross segregating population. Determination of the sexual phenotype was performed for all plants. For genotyping, a 556 bp DNA fragment of the ClACS7 gene was amplified with primers listed in S1 Table and sequenced. Polymorphisms within this fragment were used to determine recombinant alleles.

Sequence analysis

Multiple sequence alignment of full-length protein sequences of ACS was performed using the ClustalW (http://www.ebi.ac.uk/Tools/clustalw2). Phylogenetic trees were constructed by MEGA6 (http://www.megasoftware.net/index.html) based on the Neighbor-Joining method. The three-dimensional structure predictions were generated using the Geno3D server (http:// geno3d-pbil.ibcp.fr) and the visualization was carried out using the Chimera server (http:// www.cgl.ucsf.edu/chimera).

Quantitative RT-PCR experiments

Total RNA was extracted from frozen leaves, stems and flowers with the Trizol reagent (Invitrogen, France). First strand cDNA synthesis and primer design were performed as decribed in [START_REF] Boualem | A cucurbit androecy gene reveals how unisexual flowers develop and dioecy emerges[END_REF]. Primers sequences are listed in S1 Table . To check specificity of the designed primers, all amplicons were sequenced and blasted against NCBI database. Quantitative RT-PCR reactions were performed as described in [START_REF] Boualem | A cucurbit androecy gene reveals how unisexual flowers develop and dioecy emerges[END_REF]. A negative control without cDNA, technical replicates on three independent synthesis of cDNA (derived from the same RNA sample), and three independent biological experiments were performed in all cases. To compare data from different PCR runs and cDNA samples, CT values for ClACS7 were normalized to the CT value of ClActin2 (primers shown in S1 Table ). ClACS7 relative expressions were determined as described in [START_REF] Boualem | A conserved mutation in an ethylene biosynthesis enzyme leads to andromonoecy in melons[END_REF].

In situ hybridization

ClACS7 in situ hybridisation was performed as described in [START_REF] Nikovics | The balance between the MIR164A and CUC2 genes controls leaf margin serration in Arabidopsis[END_REF]. Primers used for this experiment are listed in S1 Table.

Expression and purification of the recombinant ClACS7 isoforms

The BL21(DE3)pLYSS E. coli strain {F-ompT hsdSB(rB-m rB) gal dcm (DE3) pLysS (CmR)} was used for the expression of the enzyme. The plasmid pET15b (Novagen, Germany) encodes T7 promoter, and confers ampicillin resistance. S-Adenosyl Methionine (SAM), Pyridoxal 5'phosphate (PLP) and 5'Adenylic Acid Deaminase from Aspergillus (Deaminase), were purchased from Sigma (France).

The ClACS7 enzymes expression and purification were performed as described in [START_REF] Boualem | A conserved mutation in an ethylene biosynthesis enzyme leads to andromonoecy in melons[END_REF], using BL21(DE3)pLYS cells expressing the pClACS7 constructs.

Enzyme activity assays

Enzyme activity was determined by monitoring the MTA formation by differential spectroscopy recorded on an Uvikon 940 spectrophotometer (Biotek-Kontron Instruments, France) as according to [START_REF] White | Expression of apple 1-aminocyclopropane-1-carboxylate synthase in Escherichia coli: kinetic characterization of wild-type and active-site mutant forms[END_REF]. Namely, specific activity measurement were performed on 3 different enzyme preparations by incubating 60 μM SAM in 100 mM KPhos buffer pH 8.5 (0.2 ml) and deaminase (8 μg for 0.2 ml) in the presence of 5 μM PLP in each quartz cuvette for 3 min at 25°C after addition of the purified enzyme (1 to 2 μg). The conversion of the MTA produced by ACC into an inosine derivative was monitored at 265 nm (Δε = -7740 M -1 .cm -1 ) and the specific activity was expressed as nmol of MTA formed per min per mg of protein.

Results

Cla011230/ClACS7 is the watermelon orthologue of CmACS-7/CsACS2

To test whether the genetic determinant controlling monoecy to andromonoecy sexual transition is conserved between the Citrullus and Cucumis genus and whether the watermelon ortholog of CmACS-7/CsACS2 could be encoded by the watermelon A gene, we isolated all the ACS genes from melon, cucumber and watermelon. Both cucurbit genomes contain 8 ACS genes and phylogenetic analysis determined that Cla011230, hereafter ClACS7, CmACS-7 and CsACS2 define a monophyletic clade (Fig 1A). ClACS7 encodes an ACC synthase of 444 amino acids that share 92% amino acid sequence identity with CmACS-7 and CsACS2 and all the polymorphic residues are located in non-conserved positions (S1 Fig) . Sequence analysis shows that ClACS7, CmACS-7 and CsACS2 are highly synthenic and share conserved gene structure with 3 exons and 2 introns (Fig 1B and1C). Based on this, we concluded that ClACS7 is most likely the watermelon ortholog of CmACS-7 and CsACS2. versus andromonoecy in watermelon, melon and cucumber, the high sequence identity between ClACS7, CmACS-7 and CsACS2 and the co-segregation of the A gene with ClACS7, we concluded that it is likely that the A gene encodes for ClACS7.

ClACS7 co-segregates with the watermelon andromonoecious (a) locus

Loss of ClACS7 enzymatic activity leads to andromonoecy

To understand the basis of allelic differences in the A gene, we compared the sequence of ClACS7 in 43 watermelon accessions (Fig 3) selected based on their sexual types. All tested monoecious accessions harbouring the dominant A allele presented the same ClACS7 protein sequence. In contrast, analysis of the sequence obtained from the andromonoecious accessions, harbouring the recessive a allele, revealed three missense mutations, C364W, N264K and L395F leading to two ClACS7 isoforms, C364W and N264K_L395F (Fig 3). When the closest ClACS7 homologous sequences from different plants were aligned, we observed that the residues C 364 and L 395 are conserved across seed plants whereas N 264 is located in a non-conserved region of the protein (Fig 4A) [START_REF] Ralph | Aminocyclopropane carboxylic acid synthase is a regulated step in ethylene-dependent induced conifer defense. Full-length cDNA cloning of a multigene family, differential constitutive, and wound-and insect-induced expression, and cellular and subcellular localization in spruce and Douglas fir[END_REF]. Crystallographic studies have determined that C 364 and L 395 residues are located in close proximity to the active site (Fig 4B -4D) [START_REF] Huai | Crystal structures of 1-aminocyclopropane-1-carboxylate (ACC) synthase in complex with aminoethoxyvinylglycine and pyridoxal-5'-phosphate provide new insight into catalytic mechanisms[END_REF]. The L 395 residue is located in the conserved box 7 that contains residues involved in the binding of the enzyme substrate SAM (Fig 4B -4D) [START_REF] Huai | Crystal structures of 1-aminocyclopropane-1-carboxylate (ACC) synthase in complex with aminoethoxyvinylglycine and pyridoxal-5'-phosphate provide new insight into catalytic mechanisms[END_REF]. Surprisingly, the L 395 is located at 3 amino acids of the S 399 and the S399L mutation was previously described as a natural andromonoecious m allele in cucumber [START_REF] Boualem | A conserved ethylene biosynthesis enzyme leads to andromonoecy in two cucumis species[END_REF].

To determine whether hermaphrodite flowers in andromonoecious plants are due to loss of a ClACS7 enzymatic activity, we expressed the different forms of the protein as six-histidine tag (His 6 -ClACS7) fusion proteins in Escherichia coli. The purified proteins were assessed for enzymatic activity in vitro by monitoring 5'-methylthioadenosine (MTA) formation at different PLP concentrations. The K 264 F 395 isoform was found to be totally inactive (Fig 4E and4F, Table 1). In the presence of high concentrations of PLP (100μM), the W 364 and the ClACS7 isoforms share similar biochemical constants (Table 1). However, at a physiological PLP concentration [START_REF] Boualem | A conserved mutation in an ethylene biosynthesis enzyme leads to andromonoecy in melons[END_REF], the W 364 isoform has a reduced enzymatic activity; its V max is approximately 25% of the V max of the ClACS7 isoform (Fig 4E ,Table 1). For the K 264 F 395 protein form, to determine whether the amino acid substitution of the conserved residue L 395 is responsible for the inactivation of the enzyme, we expressed and assessed the enzymatic activity of the F 395 isoform. Like the W 364 isoform, at a physiological PLP concentration, the F 395 isoform has a reduced enzymatic activity whereas at 100μM PLP, the F 395 and ClACS7 isoforms have similar biochemical constants (Fig 4E ,Table 1). The fact that the L395F amino acid substitution does not lead to a complete ClACS7 enzymatic inactivation suggests that the N264K substitution could also reduce enzymatic activity and thus the complete loss of enzymatic activity observed in the K 264 F 395 isoform is due to a synergic effect of the N264K and L395F substitutions.

Based on this and because the ClACS7 isoform from the monoecious lines was active and isoforms from the andromonoecious lines showed reduced to no enzymatic activity, we concluded that the loss of ClACS7 enzyme activity is likely the cause of the development of hermaphrodite flowers in watermelon and that ethylene production by ClACS7 is necessary for the developmental inhibition of the male organs in the female flowers of monoecious watermelon. This conclusion is reinforced by recent experiments showing that monoecious watermelons treated with an ethylene biosynthesis inhibitor, the 1-aminoethoxyvinylglycine (AVG), develop hermaphrodite flowers [START_REF] Manzano | Involvement of ethylene in sex expression and female flower development in watermelon (Citrullus lanatus)[END_REF]. Moreover, these data show that, in watermelon, the andromonoecy evolved from monoecy, independently in wild C. lanatus var. citroides accessions and in cultivated C. lanatus var. lanatus.

ClACS7 is expressed in carpel primordia of female and hermaphrodite flowers

Quantitative real-time PCR analysis demonstrates that ClACS7 is highly expressed in female and hermaphrodite flower buds and it is weak or undetectable in buds determined to develop into male flowers. No ClACS7 expression was detected in vegetative tissues (Fig 5A). To investigate in more detail the spatial expression pattern of ClACS7, we carried out in situ hybridizations. ClACS7 mRNA was detected in female and hermaphrodite flower buds at stages where buds are not morphologically distinguishable. ClACS7 mRNA was strongly localised in the carpel primordia (Fig 5C and5E). In the male flowers of the monoecious or andromonoecious watermelon lines, no ClACS7 transcript was detected (Fig 5B and5D). Since ClACS7 is expressed in both female and hermaphrodite flowers (Fig 5 ) and because the loss of ClACS7 activity accounts for the functional variation between the monoecious and andromonoecious watermelon lines (Fig 4 ), we concluded that ClACS7-mediated ethylene production in the carpel primordia prevents the development of the stamina in female flowers and is also not required for carpel development.

Moreover, since the watermelon ClACS7, the melon CmACS-7 and the cucumber CsACS2 have the same expression pattern and since the loss of ClACS7, CmACS-7 and CsACS2 enzymatic activities lead to the sexual transition from monoecy to andromonoecy, we concluded that ClACS7, CmACS-7 and CsACS2 are regulated by mechanisms common to the Citrullus and Cucumis genus and that ethylene signalling and response mechanisms leading to inhibition of stamina development in female flowers of monoecious plants are conserved in the two genera, Citrullus and Cucumis.

Discussion

Most of the species of the Cucurbitales order have unisexual flowers and are mostly monoecious (>75% of the species) bearing both male and female flowers on the same plant. Andromonoecy, a major sexual system in which plants carry both male and perfect bisexual flowers, has evolved independently numerous times [START_REF] Miller | Diversification of andromonoecy in Solanum section Lasiocarpa (Solanaceae): the roles of phenotypic plasticity and architecture[END_REF] and is found in ~4,000 species in 33 angiosperm families [START_REF] Yampolsky | Distribution of sex forms in phanerogamic flora[END_REF]. Investigations on the genetic inheritance of the different sexual morphs in the Cucurbitaceae plant family have described that the andromonoecious trait is controlled by a recessive monogenic locus [START_REF] Kubicki | Investigations on sex determination in cucumber (Cucumis sativus L.). VII. Andromonoecism and hermaphroditism[END_REF][START_REF] Nikovics | The balance between the MIR164A and CUC2 genes controls leaf margin serration in Arabidopsis[END_REF][START_REF] Poole | Interaction of sex, shape and weight genes in watermelon[END_REF][START_REF] Ji | Inheritance of sex forms in watermelon (Citrullus lanatus)[END_REF]. In our previous studies, we have demonstrated that the sexual transition from monoecy to andromonoecy, in melon and cucumber, is controlled by the common gene CmACS-7/CsACS2 [START_REF] Boualem | A conserved mutation in an ethylene biosynthesis enzyme leads to andromonoecy in melons[END_REF][START_REF] Boualem | A conserved ethylene biosynthesis enzyme leads to andromonoecy in two cucumis species[END_REF]. In this context, the genetic determinant controlling the monoecy is likely to have been established before the speciation of melon and cucumber, two species of the genus Cucumis that have diverged over 10 My ago [START_REF] Schaefer | Gourds afloat: a dated phylogeny reveals an Asian origin of the gourd family (Cucurbitaceae) and numerous oversea dispersal events[END_REF].

In this work, we described the isolation and characterization of the watermelon, Citrullus lanatus, andromonoecious (a) locus and showed that the (a) locus encodes for a rate-limiting enzyme in ethylene biosynthesis, ClACS7, the orthologous gene of the melon CmACS-7 and cucumber CsACS2. Sequence analysis of monoecious and andromonoecious watermelon accessions revealed two ClACS7 isoforms in the andromonoecious lines, the C364W and the N264K_L395F. Based on the kinetic parameters of the ClACS7 protein isoforms, we concluded that active ClACS7 enzyme leads to the development of female flowers in monoecious lines, whereas a reduction of enzymatic activity yields hermaphrodite flowers in andromonoecious lines. Expression pattern analysis showed that ClACS7 is specifically expressed in carpel primordia of female and hermaphrodite flowers and no expression was detected in male flowers and the vegetative tissues, leaves and stems. On the basis of enzymatic activity and gene expression studies and as previously described in melon and cucumber [START_REF] Boualem | A conserved mutation in an ethylene biosynthesis enzyme leads to andromonoecy in melons[END_REF][START_REF] Boualem | A conserved ethylene biosynthesis enzyme leads to andromonoecy in two cucumis species[END_REF], we concluded that ClACS7-mediated ethylene production in the carpel primordia prevents the development of the stamina in female flowers and is also not required for carpel development.

Altogether, the conserved gene structure, the genomic synteny, the conserved expression patterns and the fact that ClACS7, CmACS-7 and CsACS2 loss-of-function activities lead to hermaphrodite flower development suggest that ClACS7, CmACS-7 and CsACS2 derive from a common ancestral gene that controls monoecy before the divergence of the Citrullus and Cucumis genera, 20 My ago [START_REF] Schaefer | Gourds afloat: a dated phylogeny reveals an Asian origin of the gourd family (Cucurbitaceae) and numerous oversea dispersal events[END_REF].

Recently, a study on sex determination in squash, Cucurbita pepo, has linked the partial andromonoecy phenotype, i.e. an incomplete conversion of female into hermaphrodite flowers, to the CpACS27A gene, an ACS gene homologous to CmACS-7 and CsACS2 [START_REF] Martinez | Molecular and functional characterization of CpACS27A gene reveals its involvement in monoecy instability and other associated traits in squash (Cucurbita pepo L.)[END_REF]. The similar expression pattern in the carpels of carpel-bearing flowers (female and hermaphrodite) suggest that ClACS7, CmACS-7, CsACS2 and CpACS27A share common regulatory mechanisms along evolution and that monoecy is controlled by an ancestral gene that precedes the Cucumis, Citrullus and Cucurbita genus divergence 30 My ago [START_REF] Schaefer | Gourds afloat: a dated phylogeny reveals an Asian origin of the gourd family (Cucurbitaceae) and numerous oversea dispersal events[END_REF].

  Female flowers develop on the branches because of expression of CmACS11 that represses the expression of CmWIP1, thus releasing the expression of CmACS-7 that inhibits the stamina development. If nonfunctional CmACS-7 is expressed, hermaphrodite instead of female flowers develop. Androecious plants are obtained by loss of function of CmACS11 that leads to expression of CmWIP1 in all the flowers of the plants. Gynoecious plants are obtained by inactivation of CmWIP1 function. Hermaphrodite plants are obtained by inactivation of CmWIP1 and CmACS-7 [9-12].

  To map ClACS7 relative to the andromonoecious (a) locus, we first sequenced the ClACS7 gene in four watermelon lines, Sugar Baby, Charleston Grey, PI 296341 which are monoecious (A) harboring male and female flowers(Fig 2A and 2B), and Halep Karasi, an andromonoecious (a) line harboring male and hermaphrodite flowers (Fig 2A and 2C). PI 296341 and Halep Karasi were used to generate the mapping population. Among the four accessions, we observed 19 DNA polymorphisms in the introns and 7 in the exons (Fig 2D). Out of the 7 polymorphisms in

Fig 1 .

 1 Fig 1. Phylogeny relationship of cucurbit ACS protein family and syntenic and gene structure analysis of watermelon, ClACS7, melon, CmACS-7, and cucumber, CsACS2, genes. (A) Using the ClACS7 protein sequence as the query in tBLASTn searches, ACS protein family members were identified from watermelon, cucumber and melon. Protein sequences were aligned and used to generate the neighborjoining phylogenetic tree with 1,000 bootstrap replicates. The percentage of replicate trees in which the

Fig 2 .

 2 Fig 2. ClACS7 sequence variation and co-segregation analysis. (A-C) watermelon flower types from monoecious and andromonoecious plants. Monoecious watermelon bears male (A) and female (B) flowers whereas andromonoecious bears male (A) and hermaphrodite (C) flowers. sg, stigma; st, stamen. (D) ClACS7 sequence variation in monoecious and andromonoecious watermelon lines used in the cosegregation analysis. Polymorphic sites indicated below the gene model were positioned relative to the first nucleotide of the start codon. The red colored nucleotide indicates the C to G transition leading to the amino acid substitution C364W. Genotypes of the parental lines, PI 296341 and Halep Karasi are AAGyGy and aaGyGy, respectively. A and a genotypes on the left indicate whether the accessions harbour the dominant A or the recessive a alleles at the A locus. (E) Linkage analysis of andromonoecious (A) locus with ClACS7. The observed sexual phenotype, the number of plants, the genotype at the SNP 1477 and the genotypephenotype linkage frequency are indicated. doi:10.1371/journal.pone.0155444.g002

Fig 3 .

 3 Fig 3. Analysis of ClACS7 DNA sequence variation in 43 watermelon accessions representing all the sexual morphs of the species. Polymorphic sites indicated below the gene model were positioned relative to the first nucleotide of the start codon. The red boxes point out the nucleotide polymorphisms leading to the missense mutations, N264K, C364W and L395F leading to andromonoecious sexual morph. Presence of male, female or hermaphrodite flowers in the accessions is indicated by (x).doi:10.1371/journal.pone.0155444.g003

Fig 4 .

 4 Fig 4. Molecular characterization of ClACS7 alleles. (A) Amino acid alignments of ClACS7 isoform with homologous proteins from Cm (Cucumis melo), Cs (Cucumis sativus), Vv (Vitis vinifera), At (Arabidopsis thaliana), Le (Lycopersicum esculentum), Ph (Petunia hybrida), Zm (Zea mays) and Pg (Picea glauca). Numbers above the alignment indicate the amino acid positions along the ClACS7 protein. Box 7 indicates a conserved domain in ACS proteins. N264K, C364W and L395F amino acid polymorphisms are shown above the alignment. (B-D) Model of the three-dimensional structure of ClACS7. (B) Superposition of the tomato ACS 3D crystal structure (1IAY.pdb) indicated in blue and the 3D model of ClACS7 indicated in pink. The threedimensional structure were generated using the Geno3D server (http://geno3d-pbil.ibcp.fr) and the visualization was carried out using the Chimera server (http://www.cgl.ucsf.edu/chimera). (C and D) Zoom in on the active site of ClACS7. The residues that are polymorphic in the different ClACS7 isoforms are depicted in red. The cofactor PLP is indicated by purple ball and stick. The competitive inhibitor AVG (aminoethoxyvinylglycine) is indicated by yellow ball and stick. (E and F) Biochemical characterization of ClACS7, W 364 , F 395 and K 264 F 395 enzyme isoforms. (E) Enzymatic activity of ClACS7 (black bars), W 364 (pink bars), F 395 (orange bars) and K 264 F 395 (blue bars) protein forms. Specific activities were measured on dialyzed enzymes in the presence of 60 μM SAM and various PLP concentrations. (F) Initial velocity of the ClACS7 isoforms measured at 50 mM of PLP. Experimental data were fit into a line following Michaelis-Menten equation. doi:10.1371/journal.pone.0155444.g004

Fig 5 .

 5 Fig 5. Expression patterns of ClACS7 in watermelon flowers. (A) Quantitative RT-PCR of ClACS7 in reproductive organs, male or female flower buds from monoecious watermelon and male or hermaphrodite flower buds from andromonoecious watermelon, collected at stage 4, and in the vegetative tissues, leaves (L) and stems (S); shown are the mean ± standard deviation (s.d.) of three biological replicates. (B-E) ClACS7 in situ expression in flower buds at stages 4 in male (B) and female (C) flowers of monoecious watermelon and male (D) and hermaphrodite (E) flowers of andromonoecious watermelon. C, Carpel; St, Stamen; P, Petal; S, Sepal. Bars, 100 μm. doi:10.1371/journal.pone.0155444.g005

Table 1 .

 1 The kinetic parameters, Km and Vm, measured at 5μM and 100μM PLP for ClACS7, W 364 , K 264 F 395 and F 395 protein isoforms.

	Enzyme	5 μM PLP

PLOS ONE | DOI:10.1371/journal.pone.0155444May 12, 2016

Acknowledgments

We thank P. Audigier and N. Giovinazzo for plant care and V. Rittener for technical assistance. We thank M. Chatterjee for helpful discussions on the manuscript. We thank J. Fauve for providing watermelon andromonoecious accessions. This work was supported by BAP department in INRA, the grants Program Saclay Plant Sciences (SPS, ANR-10-LABX-40), ANR MELODY (ANR-11-BSV7-0024) and the European Research Council (ERC-SEXYPARTH).

This work was supported by BAP department in INRA, the grant Program Saclay Plant Sciences (SPS, ANR-10-LABX-40) for ABo and ABe, the Agence Nationale de la Recherche grant MELODY (ANR-11-BSV7-0024) for ABo, ABe and CD and the European Research Council grant (ERC-SEXYPARTH) for ABe. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

All relevant data are within the paper and its Supporting Information files.

Supporting Information