Confere

Mohammad Ali Mirzaei

Jean-Rémy Chardonnet

Christian Pere

Frédéric Merienne

-6 JUILLET 2012, SAN SERVOLO, VENISE TEST-BENCH FOR EVALUATING NAVIGATION AND INTERACTION IN LARGE VIRTUAL DATABASES

Keywords: 3D system, Navigation, Biological feedback, Test-bench

3D systems due to its complicated electronical, mechanical and vision accessories have enormous degree of complexity both in design and evaluation. Navigation system usually plays an important role in most 3D systems. Therefore, having an efficient navigation system and evaluation of the system has great deal of importance. 3D systems have a lot of parameters, inputs and outputs thus; evaluation process requires a complete test bench to collect and analyse maximum possible efficient parameters and variables. This paper will propose a test bench based on a tracker, navigation device status, and some biological and motion feedback from end-user to analyse a 3D system and associated navigation system performance. An original test procedure will be provided for evaluating navigation in 3D scenes by focusing on experimental aspects such as metric variable definition for measurement, case selection criteria and test-bed design. The novelty in our approach is the use of information given by the brain to understand the human perception in VE and to improve navigation in 3D scenes and interaction with virtual reality.

INTRODUCTION

With the rapid increase in performance of high-end computer graphics systems and the transition of 3D graphics onto fast and inexpensive PC platforms, virtual environment (VE) interfaces have become feasible enough to be practically used in areas such as building construction, art demonstration, industrial design, data visualization, training, and others [Gödel, 1996]. Development of useful VE applications, however, needs appropriate user interfaces in real environment and requires optimization of the most basic interactions, in particular navigation, and moving inside 3D, so that users can concentrate on high-level tasks rather than on low level motor activities [START_REF] Stanney | Realizing the full potential of virtual reality: human factors issues that could stand in the way[END_REF]. However, recently some cognitive issues have been emerged in VE such as motion sickness, and effect of different metaphors on end-users. Currently, there is little understanding of how interaction interfaces should be designed to maximize user performance in immersive virtual environments [START_REF] Mine | Virtual environment interaction techniques[END_REF]. Research that systematically investigates the human factors and design implications of immersive manipulation tasks, 3D devices, interaction metaphors and techniques remains sparse [START_REF] Stanney | Realizing the full potential of virtual reality: human factors issues that could stand in the way[END_REF][START_REF] Durlach | Virtual reality: Scientific and technological challenges[END_REF][START_REF] Brooks | Grasping reality through illusion-interactive graphics serving science[END_REF]; consequently, VE designers have had to rely on their intuition and common sense rather than on the guidance of established theory and research results. However, as Brooks [START_REF] Zhai | Human performance evaluation of manipulation schemes in virtual environments[END_REF] has noted, the uninformed and untested intuition for metaphor design is almost always wrong. Although traveling is among the most important interactions in VEs [START_REF] Herndon | The challenges of 3D interaction: a CHI'94 workshop[END_REF], we are not aware of any formal experimental studies that systematically evaluate and categorize interaction techniques for immersive traveling inside VEs. Prior research relates primarily to assessment of user performance as a function of the properties of input and output devices [START_REF] Watson | Evaluation of the effects of frame time variation on VR task performance[END_REF][START_REF] Foley | Human factors of computer graphics interaction techniques[END_REF]. In contrast, the focus of this study is on the human factor aspects of different mappings between user input (captured by input devices) and resulting actions in VEsi.e., interaction techniques [START_REF] Herndon | The challenges of 3D interaction: a CHI'94 workshop[END_REF]. A lot of interaction metaphors and their corresponding devices have been proposed during last decades [START_REF] Mine | Virtual environment interaction techniques[END_REF]; however, this paper will focus on navigation metaphors.

Travel is the motor component of the navigation task of performing the actions that move us from our current location to a new target location or in the desired direction. In addition, watching specific part of a 3D scene from different perspectives is demanding in almost all the 3D applications [START_REF] Göbel | Industrial applications of VES[END_REF][START_REF] Mine | Virtual environment interaction techniques[END_REF]. In the physical environment, travel is often a no-brainer, or to be more precise, it involves unconscious cognition. Therefore, it is quite crucial that 3D scene moves with the same (perceivable) speed as human. There are many different reasons why a user might need to perform a 3D travel task. Understanding the various types of travel tasks is important because the usability of a particular technique often depends on the task for which it is used. After a short introduction of objective system and surveillance over different parameters of the system in section 2, we will introduce a taxonomy and generic metaphor definition for navigation in VEs in section 3. We will also introduce few devices to make the metaphor application more clear. Of course, since device selection is more application dependent it is not possible to introduce a generic device. As a result just few of them will be selected to demonstrate the task definition in practice. Test-bench for evaluation will be discussed in section 4. This section will be followed by a discussion around the test bench in section 5. The paper will be ended by conclusion and references.

3D SYSTEM PARAMETES IDENTIFICATION AND MEASUREMENT

Figure 1 illustrates typical 3D environment (CAVE, car simulator, immersive room, etc.). 3D system almost will imply to 3D virtual reality in all the cases in this literature from now on. The environment consists of user in one side, 3D system at the other side, and navigation/interaction tools along with mapping interface. Therefore, three types of generic variables and parameters can be defined for this 3D environment:

-User -Navigation/interaction tools (control devices and interfacing) -3D system The movement process starts from stand still status by the user decision in the brain cortex. This signal will transmit to the navigation tool via neural system and muscular movement. After passing through mapping function the signal will be entered to the 3D system. To evaluate any interaction/navigation tools performance transfer function of navigation tools, feedback from user and 3D system are very important. Appropriate parameters and variable should be defined for evaluation in each part of 3D environment. These parameters for user, 3D system, and interfacing devices include:

User

Biological feedback such as blood pressure, heart bit, brain signal (especially vision, sensation and movement, thalamusfor cyber sickness detection), pupil diameter, acceleration and speed of the user movement and totally some multi-sensory feedback (visual, vestibular, cardiac,…) have been considered by the researchers so far.

3D systems

The speed and acceleration of the 3D scene, frame rate of the images, delay between command and task initiation, scale of a 3D model, transportation delay of the images, size (number of the points)/resolution of the 3D model, lighting conditions, colour, contrast, accuracy for positioning can be measured in the 3D system side.

Control devices and infracting

Command duration, hardware/software adjusted resolution of scene movement, position tracking errors, DOF (degrees of freedom) of control devices, calibration of device and the system, tracker placement and orientation of the tracker are the most common interfacing parameters for measurement and evaluation.

Different control devices have their own extra parameter; however they have a lot of characteristics and parameters in common. Among control devices joystick, fly-stick, Kinect, gloves, 3D mouse and keyboard are very common.

NAVIGATION AND INTERACTION METAPHORS

By definition, metaphor is the way that a selected task is done. Traveling (navigation) is one of important tasks in virtual reality. Appropriate parameters and metaphors should be taken to do these tasks. A generic traveling and tracking metaphor can be well defined as follow;

The metaphor definition shows that departure, destination points, orientation and displacement with time (specifying position, velocity and acceleration) are the very basic parameters and variables of the generic traveling.

Traveling and manipulation metaphors can be explained in more detail. For example, the following velocity/acceleration definition elaborates more details on the traveling metaphor;

Not only target and traveling parameters but also navigation device mode is also important for the interaction. For example, stop and start button is used in some devices like joystick while in Kinect base application only the human gesture has control over the movement. Albeit, more flexible control can be imposed on the traveling task in the second case than the first one.

TEST BENCH

Figure 2 shows the evaluation test-bench of a navigation/interaction system. The test-bench will provide a test procedure for evaluating navigation in 3D scenes by focusing on experimental aspects such as metric variable definition for measurement, case selection criteria and test-bed design. Performance criteria, i.e., completion time (the time taken to successfully accomplish each task) and perception time (the difference between command and end of 3D system movement) will be considered in this test-bench to develop an overall system that enables the shortest response time, thus the best navigation parameters. Subjective criteria, such as subject satisfaction, can be assessed through post-experimental questionnaires. Parameters evaluation naturally leads to take into account human factors. Thus we will analyse the effect of several human criteria such as age, gender, ethnicity and personal characteristics on overall performance and motion sickness. Through the consideration of all these parameters, we will propose a new test bench for measurement and evaluation of tracking systems (see Figure 2). In fact in this test bench stimuli from navigation device (e.g., joystick), feedback from sensation/vision cortex (electro-encephalogram signal (EEG)), accelerometer and tracker signal can be measured to compare the amount of delay between each component from the other. These time differences (delay) between each pair of signals is a good metric for evaluation of tracker systems and also deducting some biological results such as motion sickness. Velocity of 3D movement and acceleration can be calculated by following a moving object (it is not necessarily an object; the object can be a patch in the image, for example a corner or an edge) in the sequence of images that were captured by OpenGL via virtual camera. However, if we measure direct data from 3D scene by an external camera it will help us to measure real acceleration of 3D movement and velocity independent from the virtual environment. Then these results can be compared with the motion information that we have already measured from the acceleration sensor. The difference of these two measurements leads to the evaluation of motion sickness. Biological feedback plays an important role in this test bench. Since the brain signal can be used to extract information about the end user. Indeed, certain parts of the brain govern specific functions (see Figure 3). For example, the cerebellum is involved with coordination; the hippocampus with memory. Nerve cells (neurons) are the basic unit of communication in the brain. Information is relayed from one area of the brain to other areas through complex circuits of interconnected neurons. Information via electrical impulses transmitted from one neuron to many others is done through a process called "neurotransmission." The visual cortex of the brain is the part of the cerebral cortex responsible for processing visual information. It is located in the occipital lobe, in the back of the brain (yellow area).

The somatosensory system (blue area) is a diverse sensory system composed of the receptors and processing centers to produce the sensory modalities such as touch, temperature, proprioception (body position), and nociception (pain). The sensory receptors cover the skin and epithelia, skeletal muscles, bones and joints, internal organs, and the cardiovascular system. Vision and sensation region can be used to extract some feedback from the brain by EEG signal analysis. Simultaneous stimuli, brain signal, command from control module, accelerometer output can be very effective for tracking system performance evaluation since it is possible to extract the cyber sickness initiation from EEG signal. Thalamus in the brain is responsible for nausea and vomiting. EEG signal is analysed by ICA method (independent component analysis) and using brain mapping the source of signal can be localized in the brain. Thalamus condition can be continuously monitored by EEG signal. The vomiting level can be recorded by an analogue button on the control device too. In this test-bench EEG signal analysis and the user feedback can be verified with a post-experiment regular questionnaires. Many scholars have adopted a motion-sickness (MS) questionnaire by [START_REF] Chen | Spatial and temporal EEG dynamics of motion sickness[END_REF][START_REF] Kennedy | Simulator sickness questionnaire: an enhanced method for quantifying simulator sickness[END_REF] and modified for cyber-sickness to measure susceptibility of subjects to MS.

DISCUSSION

As seen in Figure 2, different measurements are saved in *.txt file with time stamp for each sample and components. For example, forward, backward, rotation to the left and right, up, down movement and their associated mechanical and software resolution are recorded for navigation tool (joystick). X, y, z coordinates and rotation around x, y and z axis are stored in the same *.txt file but in the second line. The third line is used to save measurement of the acceleration sensor at the first moment (the first time stamp). Then, the third, fourth and sixth lines of the file are used to save the over mentioned variable of navigation, tracker and sensor for the second time (second time stamp) and so on. If our sampling time is millisecond and the first index of time stamp is 0.000s then the second index is 0.001. Therefore, the second line of *.txt file is "Tracker, 0.000, x0, y0, z0, θ x0 , θ y0 , θ z0 ". A couple of images are recorded by the virtual camera of OpenGL and the external camera for each location of tracker with the same time stamp as tracker, acceleration sensor and navigation tool. For instance, the second couple of images gets 0.001 time stamp. Figure 4 shows three couples of images for three time stamps. The selected patches for calculating acceleration and velocity are highlighted by red rectangles on the images. As seen in this figure, the location of the patch was changed in the x, y axes. Three vertical lines indicate the variation in the position of the patch on the x axis, the same for y axis. The scale of the patch is changing and this proves that patch is moving along the z axis as well.

CONCLUSION

The proposed test-bench derives benefit from user, navigation tools and 3D system in order to perform more complete analysis and evaluation of a 3D environment. Acceleration and velocity of a 3D scene, user movement, tracker, and the delay between different parts of a 3D system, among all the collected data, have great influence on the final evaluation of both a 3D system and navigation tools. These variables play an important role in emerging the motion sickness while hiddenly correlated with EEG signal comes out of thalamus area. The standard post-experiment questionnaires will help to partly verify the result of EEG analysis.

Figure 1 .

 1 Figure 1. Typical 3D system and its associate components

Figure 2 .

 2 Figure 2. Navigation/interaction evaluation test-bench

Figure 3 .

 3 Figure 3. Brain map for different functions

Figure 4 .

 4 Figure 4. Recorded images with the external and internal camera


Isabelle Calves

Edouard Lavergne

Anne-Leila L Meistertzheim

Grégory Charrier

Henrique Cabral

Bruno Guinand

Louis Quiniou

Jean Laroche
email: jean.laroche@univ-brest.fr

I Calvès

Genetic structure of European flounder Platichthys flesus: effects of both the southern limit of the species' range and chemical stress

Keywords: European flounder, Estuaries, Genetics, Peripheral population, Climate change, Pollution

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

Biological factors (life history, demography) and environmental factors (oceanic hydrology, environmental stress and fishing) shape the genetic structure of marine fish populations. Recent genetic studies have demonstrated that population subdivision in marine fishes can be observed at limited geographical scales, ranging from tens to a few hundred kilometers (e.g. [START_REF] Nielsen | Genetic population structure of turbot (Scophthalmus maximus L.) supports the presence of multiple hybrid zones for marine fishes in the transition zone between the Baltic Sea and the North Sea[END_REF][START_REF] Olsen | Small-scale biocomplexity in coastal Atlantic cod supporting a Darwinian perspective on fisheries management[END_REF], despite the potential for extensive gene flow between populations in marine ecosystems. Furthermore, the adaptive significance of population structure in marine fishes is becoming evident, based on the divergence of phenotypic traits [START_REF] Hutchings | Genetic variation in life-history reaction norms in a marine fish[END_REF][START_REF] Larsen | Adaptive differences in gene expression in European flounder (Platichthys flesus)[END_REF]). Efforts to manage and conserve coastal fish species are urgently needed because this resource is particularly subject to (1) increasing impacts of global climate change [START_REF] Harley | The impacts of climate change in coastal marine systems[END_REF]) and (2) possible anthropogenic stressors at local scales; thus, the scientific community must better understand how some populations can maintain their resilience in the face of environmental degradation [START_REF] Sgro | Building evolutionary resilience for conserving biodiversity under climate change[END_REF].

Estuarine fishes displaying a wide geographical distribution and the ability to cope with contrasting environments are excellent candidates to explore evolution in populations particularly subjected to warming, chemical stress and hypoxia. In this study, we explored the genetic structure of a typical catadromous flatfish, the European flounder Platichthys flesus (Linnaeus, 1758), which spawns at sea from January to March in western Europe and has a pelagic larval life of 1 mo and a juvenile life of approximately 2 yr in estuaries. Homing behaviour was detected for the flounder in the Tamar estuary (England), where most adults return to their original estuarine range after spawning [START_REF] Dando | Site fidelity, homing and spawning migrations of flounder Platichthys flesus in the Tamar estuary, South West England[END_REF]. Finally, the European flounder is closely linked to naturally fragmented estuarine systems, allowing environmental conditions to produce noticeable phenotypic differences and possibly local adaptations.

The flounder is widely distributed in Europe from Norway to Portugal, where its abundance has decreased since the early 1990s, particularly in the Tejo estuary (Portugal). This rarefaction in Portuguese waters was associated with an increase in water temperature caused by climate change [START_REF] Cabral | Relative importance of estuarine flatfish nurseries along the Portuguese coast[END_REF]), and at the moment, the distribution of Platichthys flesus in Portugal is limited to the north and central coast. In a northern region, the Bay of Biscay, the negative impact of warming on abundance and occurrence of boreal flatfish species was also clearly detected from 1987 to 2006; indeed, the abundance of northern temperate species, like P. flesus, decreased in this region and was negatively correlated with temperatures in their year of birth [START_REF] Hermant | Impact of warming on abundance and occurrence of flatfish populations in the Bay of Biscay (France)[END_REF].

Several investigations on the genetic structure of the European flounder have been conducted over the western and northern European coasts, from Finland to Portugal.

Regarding allozymes, [START_REF] Belkhir | GENETIX 4.05, logiciel sous Windows TM pour la génétique des populations. Laboratoire Génome, Populations, Interactions[END_REF] observed (1) a significant but reduced geographic differentiation and (2) a weak pattern of isolation by distance, from the south-western Baltic Sea to southern Portugal. On the other hand, no clear indication of mtDNA differentiation was detected in that study. Using microsatellites on an extensive data set of flounder populations (from Finland to the Bay of Biscay, including western and eastern Baltic), Hemmer-Hansen et al. (2007a) detected a reduced allelic diversity in the extreme parts of the distribution area, and a higher genetic structure than [START_REF] Belkhir | GENETIX 4.05, logiciel sous Windows TM pour la génétique des populations. Laboratoire Génome, Populations, Interactions[END_REF]; they identified several barriers restricting gene flow between Trondheim and Lake Pulmanki, and between Trondheim and the North Sea, and several relatively isolated populations (Faroe Islands, eastern Baltic). In the previous study (Hemmer-Hansen et al., 2007a), the geographical distance was significantly associated with genetic divergence among the subset of samples, excluding the more isolated populations; the authors suggested a potential association between latitude and genetic structuring among the populations in the Atlantic, with the temperature gradient along latitudinal clines being the probable cause for generating such genetic structure.

Other population genetics investigations on the European flounder suggested that selective pressure due to contamination of estuaries could induce adaptive responses of the populations.

Hence, [START_REF] Marchand | Responses of European flounder Platichthys flesus populations to contamination in different estuaries along the Atlantic coast of France[END_REF][START_REF] Marchand | Physiological cost of tolerance to toxicants in the European flounder Platichthys flesus, along the French Atlantic coast[END_REF][START_REF] Marchand | Genetic polymorphism and its potential relation to environmental stress in five populations of the European flounder Platichthys flesus, along the French Atlantic coast[END_REF] emphasised an increase in the frequency of particular alleles or genotypes displaying a better capacity to cope with chemical stress, but these authors also demonstrated a physiological cost of tolerance to toxicants. In 2 other studies on Platichthys flesus, Hemmer-Hansen et al. (2007b) and [START_REF] Larsen | Adaptive differences in gene expression in European flounder (Platichthys flesus)[END_REF] confirmed the great capacity of this species to adapt to contrasting environments. These last 2 studies highlighted the genetic basis of the adaptation of flounder to the particular environment of the Baltic Sea compared to the North Sea (reduced salinity, more or less chronic hypoxia, highly variable temperature), by analysing the polymorphism of 1 candidate gene (Hsc70) and using microarray analysis.

In the present study, we analysed the genetic structure of Platichthys flesus in environmentally contrasting estuaries located along a latitudinal cline from England to Portugal (i.e. the southern limit of the distribution area of this northern temperate species).

Five estuaries were chosen because of the levels of contaminants, the possibility of hypoxia and the latitudinal cline. Our main objective was to explore the genetic structure of European flounder populations faced with climate change and anthropogenic stressors, using 3 kinds of genetic markers:

(1) A mitochondrial marker, cytochrome c oxidase subunit 1 (CO1), considered to be selectively neutral: the relative haplotype frequencies of CO1 in different populations were used to infer historical relationships between populations; the mtDNA markers have been successfully applied in phylogeographic studies [START_REF] Avise | Molecular markers, natural history and evolution[END_REF], although it is evident that selective pressure can act on them [START_REF] Ballard | Linking the mitochondrial genotype to the organismal phenotype[END_REF];

(2) 8 microsatellites, considered to be selectively neutral: these can be useful markers for phylogeographic studies, but their high variability allowed us to explore the recent evolutionary history of populations and to look for possible genetic structure considering regional spatial scales;

(3) Finally, we explored the polymorphism of a new candidate gene, the AMP-deaminase isoform 1 (AMPD1: aminohydrolase, adenosine 5'-monophosphate deaminase, isoform 1, EC 3.5.4.6) involved in the energetic metabolism and particularly in the stabilization of the adenylate energy charge [START_REF] Chapman | Stabilization of adenylate energy charge by the adenylate reaction[END_REF][START_REF] Yoshino | In situ studies on AMP deaminase as a control of the adenylate energy charge in yeasts[END_REF] and in the control of the purine nucleotide cycle [START_REF] Lowenstein | Ammonia production in muscle and other tissues: the purine nucleotide cycle[END_REF]. AMPD1 variability was analysed in the flounder populations; our hypothesis was that possible selective pressure induced by environmental stress could affect this gene.

Materials and methods

Field collection

Juvenile Platichthys flesus (average estimated age = 9 mo, average total length = 10 cm) were trawled from September to November 2009 in 5 European estuaries located along a latitudinal cline: the Tamar (England), the Canche, Seine and Vilaine (France) and the Mondego (Portugal; Fig. 1, Table 1). Approximately 50 fish were collected in each estuary (Table 1), and during the dissection in the field, 1 fin fragment was collected from each fish and stored in 95% ethanol.

All 5 estuaries presented a latitudinal gradient of temperature and different contaminant loads. The Tamar River (basin area = 10,000 km 2 ) is impacted by a high metal contamination linked to ancient mining activities [START_REF] Pulsford | Metals and melanomacrophages in flounder, Platichthys flesus, spleen and kidney[END_REF]). The Canche (basin area = 1,274 km 2 ) is a small estuary characterized by little urban development and no industrialization [START_REF] Amara | Growth and condition indices in juvenile sole Solea solea measured to assess the quality of essential fish habitat[END_REF], so it is considered as a reference site. The Seine estuary (basin area = 78,650 km 2 ) is affected by heavy chemical stress linked to high population density, numerous industrial activities and intensive agriculture across the drainage area [START_REF] Gilliers | Recherche d'indicateurs de la qualité des écosystèmes côtiers: application aux nourriceries côtières et estuariennes des poissons plats[END_REF][START_REF] Cachot | Evidence of genotoxicity related to high PAH content of sediments in the upper part of the Seine estuary (Normandy, France)[END_REF].

The Vilaine catchment (area = 10,500 km 2 ) is mainly subjected to impacts from agricultural activities and thus to pesticide contamination [START_REF] Forget | Impact neurotoxique de contaminants (pesticides et métaux) sur un crustacé marin Tigriopus brevicornis[END_REF], SAGE Vilaine 1999). Moreover, a dam is located 12 km upstream from the mouth of the Vilaine estuary which stops the inflow of saline waters into the system; in summer months, this situation can lead to stratification of the water mass in the estuary and eutrophication, and thus to severe hypoxic crisis [START_REF] Menesguen | L'eutrophisation des eaux marines et saumâtres en Europe en particulier en France[END_REF]. The Mondego catchment (area = 6,670 km 2 ) shows a generally reduced eutrophication level, and is subject to moderate levels of toxics linked to agriculture, aquaculture and industrial activities [START_REF] França | Inter-and intra-estuarine fish assemblage variability patterns along the Portuguese coast[END_REF].

mtDNA CO1 and AMPD1 variability

Genomic DNA was extracted from fin clips using AcroPrep ™ 96-well filter plates (1 ml) with 1 µm glass fibre media (PALL® 5051), following the protocol of [START_REF] Ivanova | An inexpensive, automation-friendly protocol for recovering high-quality DNA[END_REF].

Choice of markers

A fragment of the mitochondrial CO1 gene was amplified by real time PCR (qPCR) using the primers Fish-F2 and Fish-R2 (Table 2) described by [START_REF] Ward | DNA barcoding Australia's fish species[END_REF]. These primers amplified a 689 bp region of the CO1 gene. At the same time, a qPCR amplification of a 160 bp region of the AMPD gene (accession number in GenBank: AY660016) previously described (Thebault et al. 2010) was performed using Platichthys flesus-specific primers, PfAMPD1_F and PfAMPD1_R, designed by Primer Express software (Table 2). The AMPD gene of P. flesus presents 66% homology with the humanAMPD1 gene. This gene has a size of 6370 bp from the 5 ' untranslated region to the sequence of the polyA. It contains 12 exons separated by 11 introns. We decided to work on exon 1 (160 bp), because it showed a robust polymorphism in previous work on flounder populations, with 4 genotypes identified by single-strand conformation polymorphism in the Seine and Vilaine populations (Meistertzheim pers. obs.).

PCR for high-resolution melting (HRM) analysis

Real time PCR reactions were carried out in 96-well plates on a Light Cycler TM 480 Instrument (Roche) with the primers mentioned in the previous section. The reactions were performed in a 15 µl volume containing the HRM master mix (Roche) using the Resolight® set of fluorescent dyes, 3.0 mM MgCl 2 , 0.5 µM of each primer and 15 ng of DNA. Reaction mixtures were overlaid with mineral oil to prevent evaporative losses ensuring melting profile uniformity. The amplification protocol consisted of a 10 min pre-incubation at 95°C, followed by 40 cycles using the following conditions: denaturing step at 95°C for 15 s (ramp rate, 4.4°C s -1 ), touchdown annealing from 65 to 53°C for 15 s (ramp rate, 2.2°C s -1 ) and extension at 72°C for 20 s (ramp rate, 4.4°C s -1 ). The HRM genotyping method included in the LightCycler 480 v. 1.5.0 software was used to analyse the PCR products based on the melting curves. Prior to obtaining HRM curves, the PCR products were incubated at 95°C for 1 min, followed by a hybridization step of 40°C for 1 min. Melting curves were then generated by ramping from 65 to 95°C at 0.02°C s -1 , taking 25 acquisitions per each degree centigrade.

HRM curves were plotted using the automated grouping option in the LightCycler 480 v. 1.5.0 software. This protocol detects 100% of heterozygous and 75% of homozygous variants.

Homozygous variants were detected a second time at 100% by adding wild-type reference DNA in the tube as described previously [START_REF] Meistertzheim | High Resolution Melting Analysis for fast and cheap polymorphism screening of marine populations[END_REF].

Identification of variants

The Gene Scanning module of the LightCycler 480 v. 1.5.0 software was designed to improve the discrimination ability between variants. For this purpose, melting curve data were manually adjusted and fluorescence-normalized conditions were established [START_REF] Meistertzheim | High Resolution Melting Analysis for fast and cheap polymorphism screening of marine populations[END_REF]. Clustering obtained by HRM was compared with the PCR fragment sequence. When all variants had been obtained, a sample of every variant was sequenced to verify the sequence associated with each variant. Thus, 21 variants identified for the CO1 gene as well as 25 variants identified for the AMPD1 gene were sequenced.

DNA sequencing

DNA fragments amplified by real time PCR were cleaned using a PCRµ96 Filter Plate (Millipore) according to the manufacturer's protocol, eluted in 20 µl of MilliQ water and sequenced. Sequencing reactions were performed on purified PCR products with the use of the sequencing reagent BigDye V3.1 (Applied Biosystems) and the forward primer previously used for the qPCR assay. The initial phase of denaturation (2 min at 96°C) was followed by 40 cycles of 96°C for 30 s, 50°C for 30 s and 60°C for 4 min. The sequences were generated on an ABI 3730 automated sequencer (Applied Biosystems).

Microsatellite variability

First, 30 previously published Platichthys flesus microsatellites were tested [START_REF] Dixon | Molecular genetic studies of pollutant response in the European flounder[END_REF][START_REF] Casas | Characterization of microsatellite markers derived from sequence databases for the European flounder (Platichthys flesus)[END_REF][START_REF] Tysklind | Isolation and characterization of 28 new microsatellite markers for European flounder (Platchithys flesus L.)[END_REF]). PCRs were conducted on a GeneAmp PCR system 9700 (Applied Biosystems ™ ) and optimized for each locus on 8 randomly selected samples using specific published primers (forward primers were modified with the addition of a universal tail at the 5' end) and fluorescent dye-labelled universal primers [START_REF] Schuelke | An economic method for the fluorescent labeling of PCR fragments[END_REF][START_REF] Chang | Branchio-oto-renal syndrome: the mutation spectrum in EYA1 and its phenotypic consequences[END_REF]. PCR products were analysed on 1.5% agarose gels stained with ethidium bromide. Electrophoresis was performed at 130 V for approximately 30 min in 0.5% Tris-Borate-EDTA (TBE) buffer and then visualized under UV light. Only loci with clearly defined bands were retained for subsequent steps. For each selected locus, microsatellite alleles were then visualized using an ABI 3130 Genetic Analyzer (Applied Biosystems™) in order to estimate the allele size range and detect potential stuttering. Finally, 8 polymorphic loci presenting low stutter bands were retained for this study: PFSCU1, PFSCU3, PFSCU4, PFSCU7, PFSCU8, STPFL001, STPFL004 and STPFL025 [START_REF] Dixon | Molecular genetic studies of pollutant response in the European flounder[END_REF][START_REF] Casas | Characterization of microsatellite markers derived from sequence databases for the European flounder (Platichthys flesus)[END_REF] our Table 2).

To proceed with the routine PCRs and fragment analyses, the 8 forward primers were labelled (Table 2). PCR reactions were performed in a total reaction volume of 10 l, using 1 l of 10× buffer, 0.40 µl of MgCl 2 (25 mM), 0.37 µl of a pool of dNTP (2 mM, Uptima), 0.05 l of labelled forward primer and reverse primer, 0.05 µl of Taq polymerase (5000u Uptima), 1 l of template DNA (5 ng l -1 ) and 6.18 l of MilliQ water. Touchdown PCR conditions consisted of an initial denaturing step at 94°C (3 min) followed by 5 cycles at 94°C (30 s), 54 to 49°C (30 s ; -1°C cycle -1 ) and 72°C (30 s), followed by 30 cycles at 94°C (30 s), 53°C (30 s) and 72°C (30 s). A final elongation step at 72°C (5 min) ended the PCR. PCRs were performed as simplex, and PCR products were diluted at 1/200. Three multiplex fragment analyses were performed using 1 µl of each of 3 diluted PCR products pooled together (Table 2) into 10 µl of formamide (Applied Biosystems™) and 0.2 l of a 50 to 500 bp size standard (GeneScan™-500 LIZ™) in order to score microsatellite alleles on the Genetic Analyzer. Microsatellite alleles were scored using GeneMapper® Software v. 4.0 (Applied Biosystems™).

Data analysis

Cytochrome c oxydase subunit 1 CO1 sequences of 639 bp were reconstructed using both direction sequences and aligned with the alignment function of GENEIOUS Pro 5.3.4 (Biomatters™). Haplotype (h) and nucleotide () diversity values were calculated for each location using ARLEQUIN v. 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]. The most relevant substitution model was tested with jModelTest v.0.1.1 [START_REF] Posada | jModelTest: phylogenetic model averaging[END_REF]). Among the models proposed by ARLEQUIN, the best model was either that of [START_REF] Tamura | Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees[END_REF] according to Akaike's information criterion corrected for small sample sizes (AICc) or the Kimura 2-parameter model with the criterion BIC. Both models provided equivalent results, so only Tamura-Nei genetic distances were used in the presented analyses to estimate the nucleotide diversity.

The genetic structure of Platichthys flesus was investigated along the western European coast, from England to Portugal. Relationships among haplotypes were analysed in a parsimony network estimated with TCS v.1.12 [START_REF] Clement | TCS: a computer program to estimate gene genealogies[END_REF] using the statistical parsimony procedure [START_REF] Templeton | A cladistic analysis of phenotypic associations with haplotypes inferred from restriction endonuclease mapping and DNA sequence data. III. Cladogram estimation[END_REF][START_REF] Crandall | Intraspecific phylogenetics: problems and solutions[END_REF]). Fisher's exact tests of population differentiation (F ST ) based on haplotype frequencies [START_REF] Raymond | GENEPOP (version 1.2): population genetics software for exact tests and ecumenicism[END_REF][START_REF] Duran | Genetic diversity and population structure of the commercially harvested sea urchin Paracentrotus lividus (Echinodermata, Echinoidea)[END_REF] were performed in ARLEQUIN to test the null hypothesis of random haplotype distribution among sampling locations. The significance of individual tests was tested by comparison with simulated distribution constructed from 10,000 random permutations of the original data matrix. ARLEQUIN was also used to calculate the pairwise genetic distances ( ST ) according to the model of [START_REF] Tamura | Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees[END_REF]. The significance of the genetic distances was tested by 10,000 random permutations of the original data matrix.

Corrections for multiple simultaneous tests were performed by calculating the q-value (using the R package QVALUE) of each test, which measures the minimum false discovery rate (FDR) that is incurred when calling that test significant [START_REF] Storey | A direct approach to false discovery rates[END_REF].

We performed a multidimensional scaling (MDS) analysis using the function Classical (metric) Multidimensional scaling (CMD scale) in the R [START_REF] Ihaka | R: a language for data analysis and graphics[END_REF] package stats, also known as principal coordinate analysis [START_REF] Gower | Some distance properties of latent root and vector methods used in multivariate analysis[END_REF]) on linearised pairwise FST (FST /1 -FST) [START_REF] Slatkin | A measure of population subdivision based on microsatellite allele frequencies[END_REF] to assess whether any indications for group structure could be observed. Analyses of molecular variance (AMOVA; [START_REF] Excoffier | Analysis of molecular variance inferred from metric distances among DNA haplotypes: Application to human mitochondrial DNA restriction data[END_REF] were also performed in ARLEQUIN on a linearised distance matrix to quantify the level of genetic differentiation among and within groups previously identified by MDS. We executed 16,000 permutations to guarantee having less than 1% difference with exact probability in 99% of cases [START_REF] Guo | Performing the exact test of Hardy-Weinberg proportion for multiple alleles[END_REF][START_REF] Duran | Genetic diversity and population structure of the commercially harvested sea urchin Paracentrotus lividus (Echinodermata, Echinoidea)[END_REF]). In addition, to verify isolation by distance we tested the correlation between linearised pairwise distances and geographic distances between locations (measured as the shortest distance between 2 locations along the coastline) with a Mantel test [START_REF] Mantel | The detection of disease clustering and a generalized regression approach[END_REF]) implemented in ARLEQUIN, and significance was tested by 10,000 permutations.

Microsatellites

Allele frequencies and the observed and expected heterozygosity values were calculated in GENETIX v. 4.04 (Belkhir et al. 1996(Belkhir et al. -2004)). F ST (index of genetic differentiation) and single-and multilocus F IS (indicating heterozygote deficiency/excess) were estimated [START_REF] Weir | Estimating F-statistics for the analysis of population structure[END_REF] using GENETIX v. 4.04. Deviation from the Hardy-Weinberg equilibrium (HWE; Fisher's exact test) and gametic disequilibrium (Fisher's exact test) among loci were tested using GENEPOP v.4 [START_REF] Raymond | GENEPOP (version 1.2): population genetics software for exact tests and ecumenicism[END_REF][START_REF] Rousset | Genepop'007: a complete reimplementation of the Genepop software for Windows and Linux[END_REF]). Both tests were corrected for multiple simultaneous tests by calculating the q-value of each test [START_REF] Moran | Arguments for rejecting the sequential Bonferroni in ecological studies[END_REF]. The bootstrap method was chosen as recommended by [START_REF] Storey | A direct approach to false discovery rates[END_REF] for a limited number of p values. The q values were calculated using the R package QVALUE (www.r-project.org/, [START_REF] Storey | A direct approach to false discovery rates[END_REF]. Null allele frequencies were calculated based on [START_REF] Brookfield | A simple new method for estimating null allele frequency from heterozygote deficiency[END_REF] using the program MICRO-CHECKER [START_REF] Van Oosterhout | MICRO-CHECKER: software for identifying and correcting genotyping errors in microsatellite data[END_REF]).

Allele frequencies were then used to calculate pairwise genetic distances D C (Cavalli-Sforza & Edwards 1967) using GENETIX v. 4.04. The significance of the genetic distances was tested by 10,000 permutations of individuals between populations. Correlation of genetic over geographical distances (measured as the shortest distance between 2 locations along the coastline) for all pairs of populations was tested with the Mantel permutation procedure available in GENETIX v. 4.04. To assess whether any indications for group structure could be observed, we performed an MDS analysis on the linearised pairwise genetic distances [START_REF] Gower | Some distance properties of latent root and vector methods used in multivariate analysis[END_REF].

AMP deaminase

Full sequences of the fragment of AMPD1 exon 1 were reconstructed using both direction sequences and then aligned with GENEIOUS Pro 5.3.4. As for microsatellites, allele frequencies and the observed and expected heterozygosity values were calculated in GENETIX v. 4.04; deviation from HWE (Fisher's exact test) and gametic disequilibrium (Fisher's exact test) among loci were tested using GENEPOP v. 4. Both tests were corrected for multiple simultaneous tests by calculating the q value of each test, which measures the minimum FDR as explained for the CO1 data. Allele frequencies were then used to calculate pairwise genetic distances D C (Cavalli-Sforza & Edwards 1967) using GENETIX v. 4.04 (Belkhir et al. 1996(Belkhir et al. -2004)). The significance of the genetic distances was tested by 10,000 permutations of individuals between populations. Isolation by distance was tested with the Mantel test, as described for the microsatellite data.

Results

CO1

Genetic diversity within populations

We obtained 639 bp sequences of CO1 corresponding to 21 groups identified by HRM analysis stemming from the 5 populations (Tamar, Canche, Seine, Vilaine, Mondego). Among the 21 individuals, we found a total of 4 haplotypes (CO1-Pf01, CO1-Pf02, CO1-Pf03, CO1-Pf04), indicating a small degree of polymorphism. The sequences have been deposited in the GenBank Data Base (accession numbers JN859191, JN859192, JN859193, JN859194).We found a total of 3 polymorphic sites, but all mutations resulted in synonymous substitutions (Fig. 2). The statistical parsimony procedure produced a 'star-like' network; the most frequent haplotype was located in the central position of the network and was surrounded by lowfrequency haplotypes displaying a divergence of 1 mutation. Two common haplotypes were found in each location, and 1 private haplotype was found in Vilaine (Fig. 3).

Numbers of analysed individuals (n), haplotypes (N h ) and polymorphic sites (N p ), and haplotype diversity (h) and nucleotide diversity () values per location are given in Table 3.

The haplotype diversity appeared moderate and did not display a latitudinal pattern over the data set, ranging from 0.375 to 0.508, whereas the nucleotide diversity was globally low, but higher in the northern populations (Tamar and Canche:  = 0.0008) compared to the other populations ( = 0.0006).

Genetic structure

The pairwise genetic comparisons (F ST ) revealed limited differentiation (Table 4) involving several pairs of populations (Tamar/Canche; Tamar/Vilaine; Vilaine/Mondego); however, these differentiations were non-significant. A pair of populations (Tamar/Seine) showed a significant differentiation confirmed by the exact tests based on p-values; this trend was not confirmed after the correction for multiple tests (Table 4).

The MDS analysis stressed the relative isolation of the Tamar population from the other populations (Fig. 4). The analysis of the distribution of the genetic variability between the Tamar and the group of 4 other populations, performed with an AMOVA, explained 2.95% of the total genetic variance in Platichthys flesus (F CT = 0.029, p = 0.24), confirming the reduced genetic structure over the data set.

The Mantel test revealed a lack of correlation between geographic distances and pairwise Slatkin's linearised F ST values (r = -0.48, p = 0.84).

Microsatellites

Genetic diversity within populations

The microsatellite variability of Platichthys flesus appeared to be heterogeneous, with the total number of alleles per locus ranging from 7 (PFSCU3) to 35 (STPFL001) and the mean observed heterozygosity per locus varying between 0.384 (PFSCU3) and 0.765 (STPFL004) (Table 5). For each locus, the genetic diversity observed within samples was of the same magnitude over the whole data set.

Of the 8 microsatellites screened, 5 (STPFL001, PFSCU3, PFSCU4, STPFL025 and PFSCU7) exhibited substantial departures from HWE for 1 or 2 locations (Table 5). The mean allelic richness appeared higher in the northern estuaries Tamar and Canche (8.87 < r < 9.12) relative to the other populations (7.5 < r < 8.3). The mean observed heterozygosity (H o ) was similar in the 5 estuaries (0.62 < H o < 0.67). The multilocus statistics (Table 5) detected no significant deviation from HWE, regardless of the considered estuary (multilocus F IS = 0).

The software MICRO-CHECKER did not detect a significant departure from HWE linked to null alleles.

Genetic structure

The estimation of F ST showed a significant multilocus differentiation over the entire data set ( = 0.010, p < 0.05), confirmed by the multi-locus exact test of genetic differentiation (p = 0.010; Table 6). The loci STPFL001, PFSCU3, PFSCU 4, PFSCU7, PFSCU 1 and STPFL004 displayed a significant global genetic differentiation between the populations, confirmed after the correction for multiple tests (Table 6).

Pairwise estimations of F ST showed a significant differentiation mainly between Mondego and the other estuaries, and between Vilaine and Canche (Table 7). Over the 10 pairs of populations considered, 8 pairs displayed a significant genetic differentiation, confirmed after the correction for multiple tests.

The MDS analysis confirmed the isolation of Mondego from a group of northern estuaries and stressed the particular genetic status of the Seine within this group (Fig. 5). Finally, the Mantel test highlighted a significant correlation between geographical distances and linearised  (Z = 688.695; p = 0.04), suggesting an isolation by distance of flounder populations throughout the coastal European waters.

AMPD1

Allele sequencing

First, 25 HRM variants were detected among the whole data set. After sequencing of the previous 25 HRM variants, we obtained 5 real genetic variants (AMPD1-Pf01; AMPD1-Pf02; AMPD1-Pf03; AMPD1-Pf04; AMPD1-Pf05, Appendix 1). We detected 2 polymorphic sites in positions 33 and 96 in the 160 bp sequences (5'3'). Mutations were all identified as synonymous, except for allele 1 detected in locus 96, which displayed a polymorphism resulting in a modification of the corresponding amino acid (Fig. 6).

Genetic diversity within populations

Regardless of the locus considered, 2 alleles were detected per locus and per location (Table 8). H o was quite variable between the different populations (0.31 < H o < 0.60 for locus 96 where Seine > Tamar > Mondego > Vilaine > Canche; 0.12 < H o < 0.25 for locus 33 where Vilaine > Canche > Seine > Mondego > Tamar). The 2 loci showed substantial departures from HWE (Table 8). An exact test for gametic disequilibrium was performed and showed a link between the 2 loci (p = 0.03), located on the same exon and separated by 63 bp.

Genetic structure

Weir & Cockerham's  showed a significant global multi-locus genetic differentiation over the entire data set ( = 0.021, p < 0.05), confirmed by the exact test of genetic differentiation (p = 0.018; Table 9). Considered individually, locus 96 showed a significant global  value of 0.033 confirmed by the exact test (p = 0.004) which remained significant after q value correction. On the other hand, no significant differentiation over the whole data set was detected for locus 33 (Table 9).

The following analyses of genetic differentiation were carried out considering only locus 96, because this explained the whole differentiation between populations. Pairwise Weir & Cockerham's  underlined some significant genetic structure (Table 10), with the Seine estuary being significantly differentiated from the Canche, Vilaine and Mondego estuaries.

The previous pattern was confirmed by the exact pairwise test of genetic differentiation and remained significant after q-value correction (Table 10). The MDS analysis confirmed the particular genetic status of the Seine compared to a group of estuaries composed of the Vilaine, Canche and Mondego; the Tamar estuary was located between the Seine and the previous group (Fig. 7). The Mantel test did not show any correlation with geographical distances (Z = 30806.543, p > 0.05).

Discussion

Phylogeography of Platichthys flesus

The mtDNA loci are generally considered appropriate markers for inferring historical processes, thus allowing researchers to explore the phylogeography of numerous marine species widely distributed along the European coasts [START_REF] Gysels | Phylogeography of the common goby, Pomatoschistus microps, with particular emphasis on the colonization of the Mediterranean and the North Sea[END_REF][START_REF] Roman | A global invader at home: population structure of the green crab Carcinus maenas, in Europe[END_REF][START_REF] Larmuseau | Mito-nuclear discordance in the degree of population differentiation in a marine goby[END_REF]. In this study on Platichthys flesus, the analysis of mtDNA sequences identified from England to Portugal revealed (1) moderate haplotype diversity and reduced nucleotide diversity over the whole data set, and (2) 'star-like'-haplotype networks. This shallow population genetic architecture has classically been observed for numerous fish species with respect to mtDNA markers, and is probably linked to catastrophic reductions in population size or local extinctions during the Pleistocene glaciation, followed by a postglacial range expansion [START_REF] Grant | Spatial and temporal scales of genetic variability in marine and anadromous species: implications for fisheries oceanography[END_REF].

Furthermore, in this study, (1) the nucleotide diversity of the flounder was moderately higher for the northern populations (Tamar, Canche) compared to the southern group (Seine, Vilaine, Mondego), and (2) genetic differentiation between populations was very reduced, confirming previous results obtained on flounder mtDNA in the same area [START_REF] Belkhir | GENETIX 4.05, logiciel sous Windows TM pour la génétique des populations. Laboratoire Génome, Populations, Interactions[END_REF]); however, we observed a relative isolation of the Tamar estuary compared to the other sites.

The second type of genetic marker used in this work (microsatellites) can also be useful for phylogeographic studies and particularly for inferences on history of populations and possible founder events. Thus in this work, we confirmed higher genetic diversity for the Tamar and Canche compared to other populations, considering the allelic richness of microsatellites. A convergent result was obtained by Hemmer-Hansen et al. (2007a), who detected higher flounder microsatellite diversity in the North Sea and declining diversity in the extreme parts of the sampling area.

In the phylogeography of European marine fishes, it is commonly accepted that the British and Irish ice sheet extended to around 52° N at the time of the last glacial maximum (LGM: 22,000 yr BP), and that possible refugia were located in the southern North Sea and alongside unglaciated regions of the southern British Isles [START_REF] Finnegan | The genetic structuring of Atlantic salmon (Salmo salar) populations in northwest Europe as revealed through nuclear microsatellite and mtDNA PCR-RFLP analysis: from regional to catchment level analysis and its application in conservation and management of the species[END_REF]. We suggest that the southern British Isles were major refugia for flounder during the LGM and thus could explain the higher genetic variability detected in the Tamar and Canche in terms of mtDNA and microsatellites. During the deglaciation period, the colonization of new habitats towards the north and south could be associated with founder events, and thus could have led to reduced genetic variability.

Genetic structure of Platichthys flesus populations

The microsatellites used in this study are generally considered as neutral markers subjected to high mutation rates, and thus are more appropriate than mtDNA for investigating recent events which contributed particularly to the genetic divergence of populations during the postglacial period [START_REF] Grant | Spatial and temporal scales of genetic variability in marine and anadromous species: implications for fisheries oceanography[END_REF].

In the present study, a low number of significant deficits of heterozygotes was observed for particular microsatellite loci. The problem of a heterozygote deficit for microsatellite loci is well documented in marine fish [START_REF] O'connell | Microsatellite DNA in fishes[END_REF][START_REF] Karlsson | Deviations from Hardy-Weinberg equilibrium, and temporal instability in allele frequencies at microsatellite loci in a local population of Atlantic cod[END_REF].

Although factors such as inbreeding, the Wahlund effect, homoplasy or selection can explain deficits, they are frequently linked to an artifact of the PCR amplification process, i.e. to the presence of null alleles. The software MICRO-CHECKER was tested on the flounder data set and did not detect a significant departure from HWE linked to null alleles. Furthermore, in this study, the flounder populations were considered to be at HWE, thus reflecting the equilibrium between migration versus drift, classically observed for neutral markers.

The genetic variability (multilocus allelic richness and heterozygosity) was identical in the southern flounder populations of the data set (Seine, Vilaine, Mondego). Thus, genetic variability of the population was apparently maintained at the southern limit of the distribution area for the flounder (the Mondego estuary in Portugal). The flounder was clearly affected by global warming over the European coasts, thus for the period 1978 to 1997, clear changes were detected in the density of Platichthys flesus, which decreased in the Tagus estuary and, in contrast, increased in the northern Elbe estuary [START_REF] Thiel | Composition, temporal changes and ecological guild classification of the ichthyofaunas of large European estuariesa comparison between the Tagus (Portugal) and the Elbe (Germany)[END_REF]; furthermore, the abundance of flounder has decreased to extremely rare since the early 1990s in the Tagus, and its distribution in Portugal is now limited to the north and central coasts [START_REF] Cabral | Does the Tagus estuary fish community reflect environmental changes?[END_REF][START_REF] Cabral | Relative importance of estuarine flatfish nurseries along the Portuguese coast[END_REF]. The Mondego estuary is located 150 km north of the Tagus and, at the moment, probably harbours one of the most southern functional flounder populations in Europe.

In a review on the clinal patterns of genetic variation across species ranges, [START_REF] Hardie | Evolutionary ecology at the extremes of species ranges[END_REF] showed that most studies are consistent with the general perception that peripheral populations are less genetically variable than those inhabiting central areas; this decrease is particularly associated with stochastic processes (founder effects, genetic drift, isolation and/or low gene flow) which occur in marine and freshwater environments.

We suggest that the current maintenance of the genetic diversity of flounder in the Mondego could be linked to the absence of frequent demographic bottlenecks in this population and thus to a limited possibility of genetic drift or inbreeding leading to a loss of genetic variability. Furthermore, the recent rarefaction of the flounder along the coast of Portugal, and the fact that the Mondego population could be related by gene flow to northern populations less exposed to thermal stress [START_REF] Morais | The migration patterns of the European flounder Platichthys flesus (Linnaeus, 1758) (Pleuronectidae, Pisces) at the southern limit of its distribution range: ecological implications and fishery management[END_REF]), could delay the potential impact of global warming on the demography and the genetic variability of this peripheral population.

Over the sampled area, from England to Portugal, we detected moderate but significant genetic differentiation between the populations, based on microsatellite variability (F ST = 0.01 with p < 0.001). This level of genetic differentiation is comparable to that detected by Hemmer-Hansen et al. (2007a) for flounder from Norway to the Bay of Biscay; these authors occasionally observed higher levels of differentiation (0.03 < F ST < 0.09), but only for comparisons involving the highly isolated populations of the data set (Faroe Islands, Lake Pulmanki) or populations reflecting particular adaptations to brackish environments (i.e. the benthic spawners in the inner Baltic Sea). Based on the pairwise exact test of genetic differentiation in our study, we confirmed that most of the differences were statistically significant, as previously shown by Hemmer-Hansen et al. (2007a) over northern populations.

The genetic structure detected for the flounder is thus moderate but significant and robust over the whole distribution area. This differentiation between populations is probably related to the specificity of the flounder biological cycle, including (1) spawning at sea in moderate water depths (20 to 50 m), sometimes at the mouth of the estuaries, (2) a trend towards homing back to the natal spawning site, and (3) a 2 yr juvenile stage within the estuaries [START_REF] Dando | Site fidelity, homing and spawning migrations of flounder Platichthys flesus in the Tamar estuary, South West England[END_REF] where diversifying selection can operate in various environmental contexts. On the other hand, the genetic structure of a species phylogenetically close to the flounder, the plaice Pleuronectes platessa, appeared very reduced over the same area (from Norway to the Bay of Biscay), with no differentiation detected between the Irish Sea, the Baltic Sea and the majority of the North Sea samples [START_REF] Was | Microsatellite analysis of plaice (Pleuronectes platessa L.) in the NE Atlantic: weak genetic structuring in a milieu of high gene flow[END_REF]); this result is mainly related to the less pronounced links of the plaice to estuarine systems and possibly to a lack of natal homing. This study on flounder genetics was conducted in the south of the distribution area and underlines the particular genetic status of the southern population: the Mondego (pairwise differentiation involving Mondego: 0.014 < F ST < 0.027). Geographical distance is probably not a major structuring factor for the flounder populations, as the Mantel test was significant over the whole data set, but became non-significant among the subset of samples excluding the Mondego. Our results agree with those of Hemmer-Hansen et al. (2007a) for the flounder where the geographical distance was significantly associated with genetic distances but was lost when corrected for latitudinal differences between samples.

We suggest that the flounder's strict links with estuaries or freshwaters at the juvenile stage, its limited average annual migration distance (<32 km, Summers 1979) and its reduced ability to migrate over deep seas [START_REF] Aro | A review of fish migration patterns in the Baltic[END_REF]) could be the major factors structuring populations over the distribution area. Between England and Portugal, a major break in flounder habitat could be suggested in the southern Bay of Biscay linked to (1) the Capbreton canyon [START_REF] Gonthier | Instabilities and deformation in the sedimentary cover on the upper slope of the southern Aquitaine continental margin, north of the Capbreton canyon (Bay of Biscay)[END_REF]) and (2) the limited capacity of the Basque Country estuaries as nursery areas. Furthermore, a significant increase in the sea surface temperature in the southern Bay of Biscay for the period 1972 to 1993 [START_REF] Désaunay | Changes in occurrence and abundance of northern/southern flatfishes over a 20-year period in a coastal nursery area (Bay of Vilaine) and on the eastern continental shelf of the Bay of Biscay[END_REF], leading to a severe reduction in the abundance of flounder in this zone [START_REF] Hermant | Impact of warming on abundance and occurrence of flatfish populations in the Bay of Biscay (France)[END_REF], could contribute to the reduction in gene flow between the Bay of Biscay and Portugal.

Possible selective pressure on flounder populations

In this study, we considered the characterization of a partial genomic sequence of the white muscle AMPD in Platichthys flesus, revealing a high degree of conservation of both sequence and structural organization in comparison to the sequence of Rattus norvegicus [START_REF] Thébault | Partial characterization of the gene encoding myoadenylate deaminase from the teleost fish Platichthys flesus[END_REF]. We explored the polymorphism of exon 1 (160 bp) of the AMPD1 which codes for an important energy-related enzyme, involved in the stabilization of the adenylate energy charge. We detected 2 single nucleotide polymorphisms on the AMPD1 exon in positions 33 and 96, with each locus displaying 2 alleles shared by the different populations.

Contrasting levels of heterozygosity were detected for locus 96 in the whole data set; heterozygosity varied from 0.60 to 0.31, with the following classification: Seine > Tamar > Mondego > Vilaine > Canche. We observed that the first 4 estuaries displayed an excess of heterozygotes, whereas the Canche was at HWE. The previous ranking of the estuaries could be linked to a decreasing gradient of contamination. The Seine experiences diffuse contamination similar to that reported in heavily polluted estuaries of North America [START_REF] Munschy | Caractérisation chimique de la contamination organique dans l'estuaire de la Seine[END_REF], and the Tamar displays high metal contamination linked to ancient mining activities [START_REF] Pulsford | Metals and melanomacrophages in flounder, Platichthys flesus, spleen and kidney[END_REF]). The Mondego and the Vilaine estuaries show moderate levels of organic chemicals (polychlorinated biphenyls and polycyclic aromatic hydrocarbons), and seasonal high level of pesticides as a result of intensive agriculture [START_REF] Evrard | Responses of the European flounder Platichthys flesus to the chemical stress in estuaries: load of contaminants, gene expression, cellular impact and growth rate[END_REF][START_REF] França | Inter-and intra-estuarine fish assemblage variability patterns along the Portuguese coast[END_REF]. The Canche estuary is considered to be a pristine system, characterized by low levels of organic and metal pollutants [START_REF] Amara | Growth and condition indices in juvenile sole Solea solea measured to assess the quality of essential fish habitat[END_REF]. We suggest that this increasing heterozygosity for locus 96 in the flounder populations, following the contamination gradient, could be the result of selective pressure induced by the contaminants on this locus or on a linked locus. Contrasting levels of heterozygosity were also detected in flounder populations for locus 33 (0.12 < Ho < 0.25), although we are unable to formulate a hypothesis on the observed population ranking: Vilaine > Canche > Seine > Mondego > Tamar.

A significant multilocus level of genetic differentiation was detected in the whole data set.

A significant linkage disequilibrium was observed between the 2 loci, and thus we analysed the genetic structure linked to locus 96 which mainly contributed to the genetic structure; the differentiation linked to locus 33 was not significant. Significant and relatively high values of genetic differentiation were observed based on the pairwise estimations involving the Seine estuary (0.015 < F ST < 0.101). The MDS diagram showed growing genetic distances between the following pairs of populations, Seine/Tamar, Seine/Mondego and Seine/Vilaine-Canche, confirming the population ranks based on the heterozygosity of locus 96.

Finally, over the whole data set, we observed discordant patterns of variation considering the candidate gene, the AMPD1 locus 96 and the neutral markers (microsatellites). These results could indicate that selective forces have shaped the genetic population structure by given the polymorphism of the AMPD1, the genetic structure explained by the microsatellites being suggestive of stochastic forces (founder effects, migration and genetic drift). In fact, we suggest that the genetic differentiation of AMPD1 locus 96 potentially reflects the existence of cocktails of pollutants acting as selective agents in chronically contaminated populations in estuaries; we propose the hypothesis of a possible 'resistant character' associated with the heterozygosity of this locus in contaminated systems. The polymorphism observed at locus 96 showed different mutations, with 1 mutation leading to an amino acid change; we suggest that the selective pressure could act directly on this locus or indirectly through the hitch-hiking phenomenon.

This study confirms previous work showing that selective pressure of contrasting environments (variable levels of contaminants, reduced salinities, more or less chronic hypoxia, highly variable temperature) could potentially induce adaptive responses of the flounder populations [START_REF] Marchand | Physiological cost of tolerance to toxicants in the European flounder Platichthys flesus, along the French Atlantic coast[END_REF], 2010, Hemmer-Hansen et al. 2007b). However, the hypothesis of selective pressure induced by the contaminants and acting on locus AMPD1 should be confirmed in the near future, considering genotype-phenotype couplings in the field; such explorations were conducted in the past on flounder populations where differential tolerances to chemical stress were detected among genotypes [START_REF] Marchand | Physiological cost of tolerance to toxicants in the European flounder Platichthys flesus, along the French Atlantic coast[END_REF].

Perspectives

This study incorporating a phylogeographical framework and a population genetics approach based on neutral markers and a candidate gene confirms that the European flounder is probably an excellent candidate species to explore the adaptive responses of fish populations facing climate change and anthropogenic stressors in southern Europe.

Genetic monitoring of southern fish populations directly subjected to global warming must be conducted on a finer regional scale [START_REF] Schwartz | Genetic monitoring as a promising tool for conservation and management[END_REF]) and should explore how connectivity between populations could be altered by changes in ocean temperature [START_REF] Kelly | Southern hospitality: a latitudinal gradient in gene flow in the marine environment[END_REF][START_REF] O'connor | Temperature control of larval dispersal and the implications for marine ecology, evolution, and conservation[END_REF]).

If the neutral genetic diversity appears not to be reduced at the moment in the southern flounder populations, surveys must be developed to monitor population abundance and contemporary effective population size for early detection of population declines in this area [START_REF] Waples | Linkage disequilibrium estimates of contemporary Ne using highly variable genetic markers: a largely untapped resource for applied conservation and evolution[END_REF][START_REF] Antao | Early detection of population declines: high power of genetic monitoring using effective population size estimators[END_REF].

The analysis of the variability of several traits (e.g. gene expression, physiological responses and life history traits) across environmental clines should yield important information; in particular, common garden experiments can help to distinguish genetic responses (potentially adaptive) from environmental effects and phenotype plasticity [START_REF] Larsen | Adaptive differences in gene expression in European flounder (Platichthys flesus)[END_REF][START_REF] Hardie | Evolutionary ecology at the extremes of species ranges[END_REF]. Thus, we are currently conducting a common garden experiment on flounder to explore the possibility that peripheral populations compared to core populations may be differentially adapted for survival in harsh environments.

Figure 1 :Figure 2 :

 12 Figure 1: Platichthys flesus. Sampling locations of European flounder

Figure 3 :Figure 4 :Figure 6 :

 346 Figure 3: Platichthys flesus. Haplotype network of cytochrome c oxidase subunit 1 (CO1) constructed with the parsimony method of Templeton et al. (1992) for 5 populations of European flounder

Figure 7 :

 7 Figure 7: Platichthys flesus. Multi-dimensional scaling (MDS) on pairwise F ST (AMP-desaminase isoform 1, AMPD1)

Table 1 . Platichthys flesus. Fish sampling sites and number of individuals analysed for each genetic marker. CO1: cytochrome c oxidase subunit 1, AMPD1: AMP-deaminase isoform 1

 1

	Collection site	Sampling date	Microsatellites	CO1	AMPD1
	Tamar (England)	Nov 2009	43	44	41
	Seine (France)	Oct 2009	42	50	46
	Canche (France)	Nov 2009	48	50	46
	Vilaine (France)	Oct 2009	44	44	40
	Mondego (Portugal)	Sep 2009	47	46	48

Table 2 . Primer sequences and annealing temperatures for all primers used in the study

 2

	Primer	Sequence	Annealing temp. (°C) Multiplex
	STPFL001_F	5' CATCAAAGCATGAAACCC 3'	54 to 49
	STPFL001_R	5' CTGGCCCAAGTGGAGCAT 3'	54 to 49
	PFSCU4_F	5' TGGGCCGACGTGAAATAGT 3'	54 to 49	Multiplex 1
	PFSCU4_R	5' TGCTACTGGCTTTGTGATTT 3'	54 to 49
	STPFL004_F	5' ATGAGGACGTGGATGTTCTTC 3'	54 to 49
	STPFL004_R	5' CCCCTATCTCTGCTTAATGTTCAC 3'	54 to 49
	PFSCU8_F	5' ACCGCAGTTCATTGTTTGTG 3'	54 to 49
	PFSCU8_R	5' TTGGAGGGTTCCATTCTGTG 3'	54 to 49
	PFSCU3_F	5' CAGTGTAATGACCACACACCA 3'	54 to 49	Multiplex 2
	PFSCU3_R	5' CTGGACAAATAAGCATCCAAG 3'	54 to 49
	STPFL025_F	5' TTCAACAATGACCAAGGGGCATCA 3'	54 to 49
	STPFL025_R	5' TTTGCAATCTTGTAACACCTGAGA 3'	54 to 49
	PFSCU1_F	5' CTATCATGTCCCAGGGTGAA 3'	54 to 49
	PFSCU1_R	5' AGATGGAGGAGGTGGAGGAG 3'	54 to 49	Multiplex 3
	PFSCU7_F	5' GGCTCTACCCAACACCCATA 3'	54 to 49
	PFSCU7_R	5' CCATCACCTTGCTCTACTCAAG 3'	54 to 49
	Universal 1	5' TCGGATAGCTAGTCGT 3'	54 to 49
	Universal 2	5' CTGGCCGTCGTTTTAC 3'	54 to 49
	Universal 3	5' TAGTCGACGACCGTTA 3'	54 to 49
	Fish-F2	5' TCGACTAATCATAAAGATATCGGCAC 3'	65 to 53
	Fish-R2	5' ACTTCAGGGTGACCGAAGAATCAGAA 3'	65 to 53
	PfAMPD1_F	5' ATGGACGACTTTGAGCTTCCTG 3'	65 to 53
	PfAMPD1_R	5' CACCCTTTCTCCTGACCTGG 3'	65 to 53

Table 3 . Platichthys flesus. Genetic diversity (cytochrome c oxidase subunit 1, CO1) of the studied populations, showing sample size (n), number of haplotypes (N h ), number of polymorphic sites

 3

	(N p ),

Table 4 . Platichthys flesus. Genetic differentiation (cytochrome c oxidase subunit 1, CO1) of the studied populations. Pairwise F ST (above diagonal) and p-values of the pairwise exact tests of population differentiation (below diagonal), *p < 0.05. p-values did not remain significant after q-value correction

 4

		Tamar	Canche	Seine	Vilaine	Mondego
	Tamar	-	0.035	0.035*	0.008	0.005
	Canche	0.143	-	-0.013	0.019	-0.004
	Seine	0.030	0.386	-	0.000	-0.014
	Vilaine	0.091	0.129	0.344	-	-0.016
	Mondego	0.225	0.559	0.567	0.801	-

Table 5 . Platichthys flesus. Genetic diversity (microsatellites) of the studied populations. Number of alleles (N A ), observed (H o ) and unbiased (H e ) heterozygosities and F IS for each location and each locus. F IS was estimated according to Weir & Cockerham and was tested using the Markov chain method with 5,000 iterations (*p < 0.05; ***p < 0.001). Bold indicates F IS values that remained significant after q-value correction

 5

			Locus
	Population
			STPFL001 PFSCU4 PFSCU3	STPFL025 PFSCU7 PFSCU1 PFSCU8 STPFL004 Multilocus
	Tamar	NA	20	5	3	4	7	14	6	26	9.1
		Ho	0.767	0.512	0.256	0.488	0.628	0.791	0.744	0.744	0.628
		He	0.86	0.524	0.268	0.505	0.746	0.772	0.667	0.732	0.616
		FIS	+0.108	+0.023	+0.045	+0.034	+0.160* -0.024	-0.117	-0.017	0.029
	Canche	NA	17	4	3	7	7	13	7	27	8.875
		Ho	0.708	0.583	0.479	0.708	0.708	0.75	0.792	0.646	0.641
		He	0.823	0.52	0.381	0.602	0.668	0.768	0.708	0.712	0.671
		FIS	+0.141*	-0.123	-0.260*** -0.179*	-0.061	+0.024	-0.120	+0.094	-0.037
	Seine	NA	16	3	4	5	7	12	4	20	7.9
		Ho	0.814	0.488	0.372	0.558	0.674	0.697	0.767	0.744	0.625
		He	0.872	0.475	0.368	0.571	0.62	0.699	0.652	0.806	0.639
		FIS	+0.067	-0.029* -0.026	+0.023	-0.088	0.002	-0.180	+0.078	-0.010
	Vilaine	NA	14	2	5	4	9	12	6	27	8.4
		Ho	0.795	0.682	0.364	0.568	0.613	0.75	0.545	0.841	0.638
		He	0.813	0.505	0.355	0.515	0.714	0.754	0.672	0.836	0.644
		FIS	+0.022	-0.356	-0.010	-0.104	+0.142* +0.005	+0.190	-0.006	0.001
	Mondego	NA	10	4	5	5	7	12	5	22	7.5
		Ho	0.681	0.489	0.447	0.596	0.723	0.745	0.681	0.851	0.645
		He	0.802	0.536	0.399	0.546	0.681	0.788	0.653	0.814	0.651
		FIS	+0.152*	+0.088	-0.120	-0.091	-0.064	+0.055	-0.044	-0.046	0.001
	Total NA	35	10	7	9	12	22	9	22	8.35
	Mean Ho	0.753	0.551	0.384	0.584	0.669	0.747	0.706	0.765	0.635
	Mean He	0.834	0.512	0.354	0.548	0.686	0.756	0.670	0.780	0.644

Table 8 . Platichthys flesus. Genetic diversity (AMPD1) of the studied populations. Number of alleles (N A ), observed (H o ) and unbiased (H e ) heterozygosities and F IS for each location and each locus. F IS was estimated according to Weir & Cockerham and was tested using the Markov chain method with 5,000 iterations (*p < 0.05; **p < 0.01; ***p < 0.001). Bold indicates F IS values that remained significant after q- value correction. AMPD1: AMP-desaminase isoform 1

 8

	Population	Parameters	Locus 33	AMPD1 Locus 96
	Tamar	N A	2	2
		H o	0.119	0.523
		H e	0.113	0.413
		F IS	-0.051***	-0.272***
	Canche	N A	2	2
		H o	0.163	0.306
		H e	0.151	0.290
		F IS	-0.079***	-0.057
	Seine	N A	2	2
		H o	0.155	0.600
		H e	0.145	0.480
		F IS	-0.073***	-0.252***
	Vilaine	N A	2	2
		H o	0.250	0.364
		H e	0.221	0.301
		F IS	-0.132***	-0.211***
	Mondego	N A	2	2
		H o	0.120	0.420
		H e	0.113	0.358
		F IS	-0.054***	-0.176***
		Total N A	2	2
		Mean Ho	0.16158	0.4427
		Mean He	0.14902	0.36842

Table 9 . Platichthys flesus. Genetic differentiation (AMP-desaminase isoform 1, AMPD1) of the studied populations. Single-and multi-locus global assessment of genetic differentiation between samples using: (1) F ST estimated with Weir & Cockerham 's  and tested using 10,000 permutations (*p < 0.05; **p < 0.01; ***p < 0.001) and (2) exact tests of genetic differentiation tested with the Markov chain method using 5,000 iterations (bold indicates significant q-values)

 9

		Locus 33	Locus 96	All loci
		-0.006	0.033*	0.021*
	Exact test	0.539	0.004	0.018

Table 10 . Platichthys flesus. Genetic differentiation (AMP-desaminase isoform 1, AMPD1) of the studied populations. Estimation of F ST (; above diagonal) and exact tests of genetic differentiation (below diagonal) for each pair of locations. Pairwise  values were calculated according to Weir & Cockerham and were tested using 10,000 permutations (*p < 0.05; **p < 0.01; ***p < 0.001). Pairwise exact tests were tested using the Markov chain method with 5,000 iterations (bold indicates significant q-values)

 10

		Tamar	Canche	Seine	Vilaine	Mondego
	Tamar	-	0.026*	0.015	0.021	-0.000
	Canche	0.079	-	0.101***	-0.009	-0.001
	Seine	0.198	0.001	-	0.091***	0.050***
	Vilaine	0.147	1.000	0.002	-	0.002
	Mondego	0.398	0.380	0.019	0.474	-

Acknowledgements

We thank N. Quéré for excellent technical assistance. This work was financed by the ANR programme VMCS-EVOLFISH (Paris) and by the EEC programme INTERREG IV-DIESE. Sequencing was conducted at the Molecular Laboratory of the Biodiversity and Climate Research Centre (BiK-F) based on financial support of the research funding programme 'LOEWE -Landes-Offensive zur Entwicklung Wissenschaftlich-ökonomischer Exzellenz' of Hesse's Ministry of Higher Education, Research and the Arts.