Benoît Herman
email: benoit.herman@uclouvain.be

Ali Hassan Zahraee
email: zahraee@isir.upmc.fr

Jérôme Szewczyk
email: szewczyk@isir.upmc.fr

Guillaume Morel
email: morel@isir.upmc.fr

Christophe Bourdin
email: christophe.bourdin@univmed.fr

Jean-Louis L Vercher
email: jean-louis.vercher@univmed.fr

Brice Gayet
email: brice.gayet@imm.fr

Ergonomic and Gesture Performance of Robotized Instruments for Laparoscopic Surgery

Shape and mechanical structure of instruments play a large part in the lack of ergonomics during laparoscopic surgery. Providing intra-abdominal mobility and rethinking handles design are two solutions to increase comfort and precision of gestures. Based on previous work that determined the optimal intra-abdominal kinematics, this study analyses the influence of handle design on both gesture and ergonomic performance. A virtual reality laparoscopic simulator was developed to perform an experimental comparison between two novel robotized instruments and standard ones. A group of 10 surgeons and 6 researchers in robotics carried out two representative surgical tasks with each instrument. Based on instrument and arm segments tracking, a gesture performance index and an ergonomic performance index were computed. The study demonstrates that distal mobilities combined with improved handle design and integration increase ergonomic level during laparoscopy and facilitate complex gestures.

I. INTRODUCTION

Many studies in the operating room and on pelvitrainers or simulators, along with large surveys of surgeons, demonstrated that laparoscopists work in poorly ergonomic conditions. Several reasons are pointed out: 2D monoscopic vision on screen, indirect vision of hands and unnatural line of sight, inversion and variable scaling of motions, reduced intra-abdominal mobility due to the fulcrum effect of the small incisions [START_REF] Cuschieri | Whither minimal access surgery: tribulations and expectations[END_REF]- [START_REF] Vereczkei | Ergonomic assessment of the static stress confronted by surgeons during laparoscopic cholecystectomy[END_REF].

The elongated shape and mechanical structure of standard laparoscopic instruments are also implicated in those problems [START_REF] Berguer | Surgical technology and the ergonomics of laparoscopic instruments[END_REF], [START_REF] Emam | Influence of handle design on the surgeon's upper limb movements, muscle recruitment, and fatigue during endoscopic suturing[END_REF]. The orientation of the handle (pistol-shaped) with respect to the shaft and the length of the latter increase the movement amplitude of the upper limb and the strain in the surgeon's shoulders, elbows and, above all, wrists. The unusual posture of upper limbs combined with the weak mechanical efficiency (friction) in the opening mechanism of instruments are reported to increase muscles recruitment. This overall lack of ergonomics in laparoscopy is at the source of pain and feeling of discomfort [START_REF] Berguer | Ergonomic problems associated with laparoscopic surgery[END_REF], [START_REF] Vereczkei | Ergonomic assessment of the static stress confronted by surgeons during laparoscopic cholecystectomy[END_REF], [START_REF] Nguyen | An ergonomic evaluation of surgeons' axial skeletal and upper extremity movements during laparoscopic and open surgery[END_REF]. Most of these aches are due to the increase of muscular efforts in the upper limb. Wrist and elbow tendinitis are rather common among experienced laparoscopists because of the awkward arm postures they are used to take-elbows raised and pointing outwards or even upwards, wrists bent and twisted excessively. Furthermore, neck and back stiffness and fatigue often occurs, due to the static position of the trunk and head of the surgeon who looks at the screen most of the time. Finally, most instruments are closed by thumb and the application of a high force on a small contact surface compresses one of the medial nerve endings. This often causes thumb numbness that may spread to the whole hand.

Various tracks have been followed for the last decade to improve comfort and raise the quality of laparoscopic surgery. In addition to the broad diffusion of instructions (e.g. trocars placement, table adjustment, spatial organization of monitors and surgical team) for operating in the most adequate posture, an increasing interest is taken in the development of novel instruments. In particular, industrials and researchers work on two main topics: providing additional intra-abdominal mobility by adding several joints at the distal tip of instruments, and designing more ergonomic handles [START_REF] Matern | Instruments for minimally invasive surgery: principles of ergonomic handles[END_REF]. A comprehensive review of manual and robotized articulated instruments can be found in [START_REF] Zahraee | Mechatronic design of a hand-held instrument with active trocar for laparoscopy[END_REF].

Intra-abdominal degrees of freedom (DOF) mainly intend to increase quality and precision of several gestures (e.g. blood vessels cut and coagulation, clip placement, intracorporeal stitch and knot) that are rather difficult to perform with the only 4 DOF of conventional instruments. In [START_REF] Zahraee | Toward the development of a hand-held surgical robot for laparoscopy[END_REF], we compared several 2 and 3 DOF distal kinematics through suturing trials on a virtual reality (VR) simulator that was developed for the purpose. The most appropriate kinematics (i.e. Yaw-Roll) was then implemented in several prototypes.

As a supplement to this initial study, the present paper focuses on the design of an ergonomic handle and its integration to the articulated instrument, so as to allow the surgeon to work in a more comfortable posture and to control intuitively the additional mobilities.

II. MATERIALS AND METHODS

To assess the influence of distal DOFs and handle design on gesture and ergonomic performance in laparoscopic surgery, an experimental protocol was set up. Using a VR simulator, novel instruments were compared with standard graspers through two representative tasks.

A. Instruments

The 3 instruments depicted on Fig. 1 were evaluated in this study. In addition to two robotized instruments each featured with a controllable Yaw-Roll intra-abdominal part (described below), the subjects also performed the exercises with standard instruments, in order to quantify the advantage of the new instruments over current ones.

To give the surgeon a direct and intuitive control of the additional tip mobilities, a Wii Nunchuk is used as handle. It is designed ergonomically, so the user has a good grip on it. Its 2-DOF joystick can be used to control the actuated intraabdominal motions. The Nunchuk handle can be fixed on the shaft (see Fig. 1b) with an angle that maximizes ergonomic performance and gesture quality, according to [START_REF] Ahmed | Optimal angle between instrument shaft and handle for laparoscopic bowel suturing[END_REF].

However, the intra-abdominal workspace is large. As a consequence, ergonomic level of the instantaneous posture highly depends on the intra-abdominal region where the surgeon works. Indeed, some orientations of the instrument shaft impose an awkward arm configuration with often high flexion and deviation of the wrist. To solve this problem, we introduced in [START_REF] Zahraee | Mechatronic design of a hand-held instrument with active trocar for laparoscopy[END_REF] an alternative version of the robotized instrument that comprises a free spherical joint between handle and shaft (see Fig. 1c). This passive joint releases the kinematic constraints on the wrist and allows to cover the whole intra-abdominal workspace while keeping the hand in line with the forearm.

The presence of this 3-DOF joint implies that the handle can turn freely around the instrument longitudinal axis without turning the shaft. This motion has therefore to be motorized. As detailed in [START_REF] Zahraee | Mechatronic design of a hand-held instrument with active trocar for laparoscopy[END_REF], a motor is embedded in a part of the instrument called active trocar that is tightened on a standard trocar. It allows a free manual translation of the instrument inside the trocar but controls the rotation of the shaft that cannot be performed manually. Two buttons of the Nunchuk (additionals to the joystick) are used to turn the instrument around its axis.

B. Surgical Tasks

In order to quantify the performance of laparoscopists, many studies have suggested over the last decade various protocols for evaluation of laparoscopic skills during and after training. These exercises can be performed on a pelvitrainer, a VR simulator or in vivo using animal models. Derossis et al. [START_REF] Derossis | Development of a model for training and evaluation of laparoscopic skills[END_REF] proposed a series of 7 exercises that cover the whole range of gestures performed by surgeons during any laparoscopy: grasping an object and passing it from one instrument to the other, dissecting and cutting along a prescribed trajectory, clipping and cutting a vessel, ligaturing a vessel with a thread, placing and stitching a mesh, suturing with an intra-corporeal knot, and suturing with an extracorporeal knot. Cao et al. [START_REF] Cao | Task and motion analyses in endoscopic surgery[END_REF] analyzed laparoscopic gestures from a kinematic point of view and identified 5 motions or actions: pointing and reaching a target and orienting the effector, closing the effector to grasp, hold or cut, pushing with the instrument, pulling, and opening the effector.

For practical reasons, we tried to minimize the number of tasks to perform during the experiments. We selected 2 exercises from Derossis' set: a pointing task and a pick and place task. They cover most of Cao's motions and actions, and are pretty representative of laparoscopic gestures. Furthermore, they are not too long and can be repeated several times.

Pointing task (see Fig. 2a): it consists in moving the instrument tip in free path and touching targets (blue sphere in Fig. 2a) that appear successively in the workspace. This task does not require the intra-abdominal DOFs (that are therefore disabled) and essentially focus on the handle influence on ergonomic performance and motion accuracy.

Pick and place task (see Fig. 2b-c): the user has to pick a ring and place it on a pin in another position. This is a more complex task than pointing that requires hand and wrist Furthermore, the task is designed so that the use of intraabdominal mobilities is mandatory to succeed: the ring can only be grasped on one portion (in blue on Fig. 2b). As a consequence, it cannot be accomplished with a single straight instrument. This could be seen as a bias at the advantage of articulated instruments but we believe that it depicts the reality. Indeed, to prepare a suture when using standard instruments, a surgeon has to pass several times the needle from one instrument to the other to orient it properly. Therefore, a second straight instrument must be held by the subject to carry out the pick and place task.

As it is not usual for a surgeon to operate with one hand free, the subject had to hold this second straight instrument during all trials-both tasks with each instrument-, although it was unused most of the time.

C. Metrics

Gesture performance: Satava et al. [START_REF] Satava | Metrics for objective assessment[END_REF] drew up a list of all laparoscopic skills assessment devices and metrics. It emerges from this review that gesture quality is usually quantified with a few simple metrics: time to completion, number of motions, number of errors, total distance traveled by the instrument tip, and spectrum of velocities and accelerations. A global index is also commonly used, made of a weighted sum of several metrics. We adopted a global performance index proposed by Huang et al. [START_REF] Huang | Fuzzy classification: towards evaluating performance on a surgical simulator[END_REF] that combines the time to completion (T T C), the number of errors (Err) and the motion economy (M E), defined as the ratio between actual and optimal (shortest) path length:

P erf ormance = T T C + Err + 2 × M E. (1)
The weight for motion economy was chosen in agreement with surgeons, regarding the expected values for the two other metrics. An error is recorder each time an instrument oves out of the screen (during both exercises) or when the ring collides with the target pin (pick and place task).

Ergonomic score: McAtamney and Corlett [START_REF] Mcatamney | RULA: a survey method for the investigation of work-related upper limb disorders[END_REF] proposed a method for quantifying the ergonomic level of work environment, called Rapid Upper Limb Assessment (RULA). The RULA score is calculated from the upper limb angles, general posture of trunk and neck, muscular load and time evolution of posture. The score ranges from 1 to 7, increasing when ergonomic level decreases. Person et al. [START_REF] Person | Automated high-frequency posture sampling for ergonomic assessment of laparoscopic surgery[END_REF] established a real-time ergonomic index based on RULA, adapted to laparoscopic surgery. This index takes into account the movements of both arms, the trunk and the neck and is suitable for real-time motion tracking. We developed a similar RULA-based index that takes into account only the motions of the major arm (the one that holds the main instrument). Our ergonomics score comes from Table A of the original RULA score and ranges from 1 to 9 (the lower the better). It is obtained from 4 individual posture scores detailed in I: upper-arm score, forearm score, wrist posture score and wrist twist score. These individual posture scores are computed from joint angles defined in [START_REF] Romilly | A functional task analysis and motion simulation for the development of a powered upper-limb orthosis[END_REF] and depicted in Fig. 3. Joint angles are retrieved by means of a 3D localizer (see Section II-D). A set of 7 markers are placed on the subject (see Fig. 3): both shoulders (Acromioclavicular joint), major elbow (lateral and medial epicondyles), major wrist (radial and ulnar styloids), and major hand (metacarpal end of the middle finger). Center of rotation of wrist and elbow are supposed to be the midpoint of the segment joining the two corresponding markers. Center of the shoulder can be obtained using a spherefit algorithm with successive positions of one elbow marker (expressed in a frame attached on the subject's torso). Upper limb segments are then reconstructed between successive joint centers and angles values can be

D. Experimental Setup

The global experimental setup is shown in Fig. 4. Instruments mock-ups produced by rapid prototyping were inserted in a pelvitrainer through trocars that form a 10 cm equilateral triangle with a virtual laparoscope. Viewing angle of the latter was 45°with respect to the table plane. A 19" monitor was placed at 1.5 m from the subject's eyes, with a line of sight close to 25°downwards. Table height was adjustable to fit with all subjects.

Exercises were implemented on a VR simulator. Instruments position and orientation were measured with a Polaris Optotrak system (Northern Digital Inc.) connected to the VR simulator via a serial port. This device can localize several targets with a precision of 0.3 mm, using 2 infrared (IR) cameras, IR emitters and reflective spheres on each target.

Upper limb segments were tracked using a Codamotion system (Charnwood Dynamics Ltd.), made of a Hub computer unit and 3 Cx1 units. A Cx1 unit comprises 3 IR sensors that track IR-emitting markers placed on the subject's arm. Each Cx1 unit is capable of retrieving the 3D position of all markers, but using 3 units improves accuracy (< 0.1 mm) and provides redundancy. No interference occurred between Ploaris and Codamotion localizers.

All experiments were recorded using a camcorder placed in front of the subject.

E. Protocol

Two groups of subjects were recruited for the study:

• 10 surgeons from different specialties (urology, gynecology, general and digestive surgery) with various levels of expertise in laparoscopy and surgical robotics; • 6 PhD students in robotics with no experience in laparoscopy but used to playing video games. During the placement and calibration of the Codamotion markers, the experiment purposes and methods were introduced to the subject. Emphasis was put on the fact that only instruments performance are measured and compared, with no intention to rank individuals. Subjects were told to perform each trial as quickly as possible, with the shortest path, and avoiding errors.

The experiment started with the standard laparoscopic instrument. A short demonstration was carried out to explain the pointing task. Then the subject repeated several times a learning exercise similar to the task before the recorded task itself, in order to get familiar with the VR environment, the task and the instrument. A learning curve was calculated after each learning exercise with the time and performance score and the subject could start the recorder trial only after reaching a learning plateau, to avoid learning during the trial. After the pointing trial, the same steps (demonstration, learning exercises, recorded task) were repeated for the pick and place task.

Each subject repeated this protocol for both dexterous instruments. These were used in alternate order among subjects in order to minimize the effect of learning the exercises with an instrument on the results with the other instrument.

F. Statistical Analysis

The performance score can be computed easily for each recorded trial. On the contrary, our RULA-based ergonomic score is instantaneous and computed for each Codamotion frame of a trial. The mean value of the ergonomic score (called 'RULA-mean' hereafter) was therefore calculated for each trial (as in [START_REF] Person | Automated high-frequency posture sampling for ergonomic assessment of laparoscopic surgery[END_REF]). In addition, standard deviation (RULA-SD) and minimum and maximum values (RULA-min and RULA-max) were also computed for each trial to have a more detailed view of the general ergonomic level.

For each task, an ANOVA was performed using Statistica to identify significant differences between instruments or subjects groups (students and surgeons), regarding either gesture or ergonomic performance. A complementary Newman-Keuls post-hoc test was performed when the ANOVA significance level was reached.

III. RESULTS

A. Ergonomic Score

Fig. 5a shows the mean values of the ergonomic scores (i.e. RULA-mean, RULA-SD, RULA-min, and RULA-max) computed for all trials of the pointing task, sorted by subjects group and instrument. Results for the pick and place task are summarized in Fig. 5b.

For the pointing task, the statistical analysis performed on RULA-mean does not show any significant difference between the 3 instruments. For surgeons however, the RULAmean score tends to be lower with the articulated handle, although it is not significant (p > 0.05). RULA-SD values show a significant effect of instrument (F (2.26) = 7.1330, p < 0.05), the articulated handle performing better than the other two), especially for students although there is no significant interaction between instrument and expertise. RULA-min values do not show any statistical trend. RULAmax values also show a significant effect of instrument (F (2.26) = 7.4533, p < 0.05), with higher performance for (a) Pointing task (b) Pick and place task Fig. 5. RULA-based ergonomic scores; center of the box is the mean value of RULA-mean, height of the box is the mean value of RULA-SD, and limits of the segment are mean values of RULA-min and RULA-max both robotized instruments, especially for students again but with no significant interaction.

For the pick and place task, RULA-mean values show a significant effect of instrument (F (2.28) = 17.1770, p < 0.05) and a significant interaction between instrument and expertise (F (2.28) = 11.8920, p < 0.05). The same significant trend can be observed among both groups, standard instrument having lower performance that robotized instrument with fixed handle. However, a significant difference exists between surgeons and students regarding the articulated handle, which has the best performance for students but not for surgeons. RULA-SD values show a significant effect of instrument (F (2.28) = 3.8138, p < 0.05) with articulated handle showing better performance. RULA-min values do not show any statistical trend. RULA-max values show a statistical advantage for articulated instruments for both groups (p < 0.05). There is a trend to interaction between instrument and group: the articulated handle has the best performance for students but not for surgeons.

B. Gesture Performance

Fig. 6a and6b show the mean value and standard deviation of the global performance score computed for all trials, sorted by subjects group and instrument, for the pointing and pick and place tasks respectively.

For the pointing task, the articulated handle is significantly less efficient (F (2.26) = 37.4650, p < 0.05). There is a trend to interaction with expertise (p = 0.06): the fixed Nunchuk is the best for students, but do not perform significantly better than the standard instrument for surgeons.

For the pick and place task, there is a significant effect of instrument (F (2.28) = 4.9722, p < .005) with the fixed Nunchuck performing better that the other two.

IV. DISCUSSION

From an ergonomic point of view, the novel solution of adding a free (passive) 3 DOF spherical joint between handle and shaft seems to be very effective. The instrument with the passive articulation is globally the most ergonomic one. All ergonomic criteria are clearly better with this instrument for the PhD student group. This is an interesting and encouraging result as none of the existing inner-DOF instruments has a solution to the problem of ergonomic shortage. Among surgeons, this trend is less visible and ergonomic scores are not as good as for roboticists. This can be explained by the fact that surgeons are used to working with the standard instruments: they have better ergonomic scores than novices for the complex pick and place task with such instruments. It is known in many fields (even outside medicine) that specialists experience some difficulties to replace a well known technique by a new one, as they have to unlearn their habits in order to exploit all capabilities offered by the new device. It can therefore be expected that, with an appropriate learning and training phase, surgeons will fill in the progression margin and perform as well as (or even better than) the students group.

From an efficiency point of view, the free joint handle is not the best solution among the 3 instruments. The dexterous instrument with a fixed handle is either the most efficient or as efficient as the conventional one. This is because controlling accurately the position of the end-effector is more difficult with the free joint. This clearly influences the results for the pointing task and could better be visualized through a spectral analysis of speeds and accelerations. Another disadvantage of the free handle is that it requires one more actuator-and consequently, one more control means-than with a fixed one, as explained in Section II-A. This increases the complexity of control, as we could notice during the experimental campaign. Subjects that could easily drive the 2 intra-abdominal DOF with the fixed Nunchuk became hesitant with this additional DOF. This adds up with the precision problem and explains the rather poor results during the pick and place task. However, one can notice that both groups performed better with distal mobilities during the manipulation task. This demonstrates the benefits of intraabdominal mobilities for complex surgical gestures. One surprising point is that students performed globally better than surgeons for the pick and place task, regardless the instrument. This can probably be explained by the fact that our VR simulator is rather removed form surgical reality: absence of anatomical landmarks, no perception of interaction forces between tool and tissues. This can unsettle expert surgeons a bit while PhD students in robotics consider the VR interface as a video game that they are used to learn quickly.

V. CONCLUSIONS AND FUTURE WORK

To solve a part of the lack of ergonomics in laparoscopic surgery and facilitate complex gestures, a novel instrument was designed. It comprises an articulated end-effector with Yaw-Roll intra-abdominal mobilities, and an ergonomic handle that is linked to the shaft through a passive spherical joint. Both ergonomic and gesture performance of this new instrument were assessed through a series of experiments on a VR simulator. 10 laparoscopists and 6 PhD students in robotics took part in the study.

It is shown that the additional mobilities improve performance (i.e. completion time, economy of movement) for complex tasks. The articulated handle allows for working in a more ergonomic way but requires a learning and adaptation phase to reach the same level of performance. It could be of interest to study more deeply the role of learning exercises on surgical performance with a novel instrument that modifies noticeably experts' habits. For example, the evaluation trials could be repeated several times during a 2 weeks learning and training protocol with the new instruments, and several weeks after the training phase.

It would also be worth trying to combine the advantages of the articulated handle in terms of ergonomic quality with the performance of the same instrument with a fixed handle. A solution might be to provide a handle that could be adjusted freely during large instrument repositioning and locked afterwards for performing fine gestures in a narrow workspace, with little changes in the arm posture.

Finally, the experimental setup should also be updated to offer force feedback. This seems more important than designing an anatomical environment, as most surgeons are used to train on pelvitrainer.

Fig. 1 .

 1 Fig. 1. Kinematics of the three instruments that were compared experimentally: a) standard instrument; b) robotized instrument with 2 intra-abdominal DOF and fixed handle; c) robotized instrument with 2 intra-abdominal DOF, active trocar and moving handle

Fig. 2 .

 2 Fig. 2. Pointing and pick-and-place tasks in the virtual reality simulator: a) pointing task; b) grasping during pick-and-place task and c) approaching the destination pin during pick-and-place task

Fig. 3 .

 3 Fig. 3. Definition of upper limb angles used to compute the RULA-based ergonomic score; black-filled circles are the joint centers, empty circles are the markers positions

Fig. 4 .

 4 Fig. 4. Experimental setup during a trial

Fig. 6 .

 6 Fig.6. Global performance scores; center of the segment is the mean value, and height of the segment is the standard deviation

ACKNOWLEDGEMENT

The authors would like to thank warmly all surgeons at Institut Mutualiste Montsouris and students at Institut des Systèmes Intelligents et de Robotique who took part in the experiments.

This work was supported by Agence Nationale de la Recherche grant ANR-09-CORD-020.


Carolina Camus

Sylvain Faugeron

Alejandro H Buschmann
email: abuschma@ulagos.cl

Assessment of genetic and phenotypic diversity of the giant kelp, Macrocystis pyrifera, to support breeding programs

Keywords: breeding, genetic diversity, phenotypic diversity, microsatellites

Assessment of genetic and phenotypic diversity of the giant kelp, Macrocystis pyrifera , to support breeding programs

Carolina Camus, Sylvain Faugeron, Alejandro

Introduction

The use of seaweeds for food/feed, pharmaceutics, textiles, cosmetics, and biofuels [1,2 3 4,5] and the continuously growing demand for raw material, is rapidly changing the way that we humans interact with this natural resource [START_REF] Loureiro | Seaweed cultivation: potential challenges of crop domestication at an unprecedented pace[END_REF]. Encouraged by this increasing demand and the need to reduce the over-exploitation of natural resources, seaweed farming is expanding across several continents from East Asia to Europe, South America and East Africa [START_REF] Rebours | Seaweeds: An opportunity for wealth and sustainable livelihood for coastal communities[END_REF]. Within aquaculture, the global production of seaweeds is 27.3 million tons (27%), and it has increased by 8% per year over the past decade [START_REF]Contributing to food security and nutrition for all[END_REF].

Under this scenario, seaweed farming requires the urgent development of breeding programs to increase yield and optimize other relevant agronomic traits [START_REF] Acquaah | [END_REF][START_REF] Gjedrem | Genetic improvement for the development of efficient global aquaculture: A personal opinion review[END_REF]. There is a large amount of information on the development of macroalgal strains in red and brown algae [START_REF] Li | Improving seedless kelp (Saccharina japonica) during its domestication by hybridizing gametophytes and seedling-raising from sporophytes[END_REF][START_REF] Shan | Breeding of an elite cultivar Haibao No.1 of Undaria pinnatifida (Phaeophyceae) through gametophyte clone crossing and consecutive selection[END_REF][START_REF] Yan | Selection and characterization of a hightemperature tolerant strain of Porphyra haitanensis Chang et Zheng (Baqngiales, Rhodophyta)[END_REF]. However, the genetic science behind seaweed breeding and domestication is still in an initial phase, with little conceptual and empirical progress [START_REF] Robinson | Genetic improvement of macroalgae: Status to date and needs for the future[END_REF]. Several challenges related to the biological peculiarities of algae and their environment are yet to be faced. For instance, the marine environment is more complicated to manipulate than terrestrial environments, where water and nutrient supply, ploughing and other manipulations modify the physico-chemical properties of the soil, and avoid competitors, predators and most pests and pathogens. Such manipulations are generally not possible in the sea, without significant logistical and infrastructure costs, which are sometimes accompanied by unwanted side effects [START_REF] Chung | adaptation against global warming[END_REF]. Therefore, the increase in productivity must strongly rely on the modification of heritable traits. Yet, the genetic improvement of any agronomic trait must ensure the retention of adequate genetic variance in the targeted traits in order to ensure sufficient scope for adaptation to local environmental variation. Another major difference with land plants, where current breeding efforts are concentrated in already domesticated strains, is the use of wild variants for most seaweed aquaculture initiatives [START_REF] Peteiro | Mariculture of the Asian kelp Undaria pinnatifida and the native kelp Saccharina latissima along the Atlantic coast of Southern Europe: An overview[END_REF][START_REF] Hurtado | Developments in production technology of Kappaphycus in the Philippines: more than four decades of farming[END_REF]. Some of the cultivated algal species have never gone through a selective breeding process, based on genetic knowledge. Currently, no more than eight species are in the early stages of domestication [START_REF] Valero | Perspectives on domestication research for sustainable seaweed aquaculture[END_REF]. One possible reason for this maybe the ease with which selected strains from wild populations can be cloned in order to establish a new seaweed farm. On the other hand, the complex life histories of algae, add additional conceptual and practical constraints to the implementation of breeding programs [START_REF] Valero | Perspectives on domestication research for sustainable seaweed aquaculture[END_REF]. Indeed, trait correlations among life cycle stages may have negative consequences on overall production and/or breeding efforts [START_REF] Marshall | Ecological and evolutionary consequences of linked life-history stages in the sea[END_REF]. For example, selecting for growth rate in the farmed red alga Gracilaria chilensis caused the dominance of heterozygous diploids that lost their capacity for sexual reproduction [START_REF] Guillemin | Genetic variation in wild and cultivated populations of the haploid-diploid red alga Gracilaria chilensis: How farming practices favor asexual reproduction and heterozygosity[END_REF][START_REF] Guillemin | Evidence of reproductive cost in the triphasic life history of the red alga Gracilaria chilensis (Gracilariales, Rhodophyta)[END_REF][START_REF] Guillemin | Tracing the trans-pacific evolutionary history of a domesticated seaweed (Gracilaria chilensis) with archaeological and genetic data[END_REF] and contributed to the critical loss of genetic diversity observed in this species [START_REF] Guillemin | Genetic variation in wild and cultivated populations of the haploid-diploid red alga Gracilaria chilensis: How farming practices favor asexual reproduction and heterozygosity[END_REF]. Strong genetic diversity losses in cultivated populations can have serious consequences for the adaptability of these species and their susceptibility to pests and diseases [START_REF] Loureiro | Seaweed cultivation: potential challenges of crop domestication at an unprecedented pace[END_REF][START_REF] Buschmann | Intertidal Gracilaria farming in southern Chile: differences of the algal proveniance[END_REF][START_REF] Ask | Advances in cultivation technology of commercial eucheumatoid species: A review with suggestions for future research[END_REF][START_REF] Li | Prediction of the heterosis of Laminaria hybrids with the genetic distance between their parental gametophyte clones[END_REF][START_REF] Li | Breeding and trial cultivation of Dongfang No. 3, a hybrid of Laminaria gametophyte clones with a more than intraspecific but less than interspecific relationship[END_REF][START_REF] Halling | Introduction of Asian strains and low genetic variation in farmed seaweeds: Indications for new management practices[END_REF].

One of the main challenges that seaweed-breeding science is currently facing is the lack of general knowledge on the drivers of phenotypic diversity. While a large body of literature has analysed phenotype responses to environmental variation, relatively little is known about genetic determinism of algal traits and their interactions with environmental determinants. Quantitative genetics approaches on algal models, such as QTL analysis [START_REF] Shan | De novo transcriptome analysis of the gametophyte of Undaria pinnatifida (Phaeophyceae)[END_REF][START_REF] Avia | High-density genetic map and identification of QTLs for responses to temperature and salinity stresses in the model brown alga Ectocarpus[END_REF], have recently emerged, and should provide valuable tools to assist breeding strategies in the near future . However, because most cultivated seaweeds are not yet domesticated, the production of new varieties must rely on an initial genetic pool collected from natural populations. Therefore, a critical initial stage in the establishment of a breeding program is the acquisition of solid knowledge concerning the natural variation in both the phenotypic and genotypic diversities [START_REF] Loureiro | Seaweed cultivation: potential challenges of crop domestication at an unprecedented pace[END_REF]. Several fundamental questions can be tackled from such knowledge: 1) Can selection (either natural or artificial) modify traits of interest such as growth rate or shape (among many other traits)? By investigating signatures of evolutionary divergence between environments within the species range, it is possible to infer the evolvability of the species of interest under natural conditions, which is related to the capacity of different traits to accumulate additive genetic variation. The existence of such genetic diversity is essential to the success of trait improvement by selective breeding.

2) Can new varieties be cultivated anywhere or should landraces be established? Because aquaculture systems are deeply influenced by the natural environment, which cannot be easily modified or controlled, it is likely that selected strains or wild progenitors that evolved local adaptations will not be able to grow optimally in non-native environments. In this context, breeding strategies based on selection of local variants should maintain the genetic diversity necessary for optimal growth in the farm environment to secure the sustainability of the production. 3) Should breeding strategy be oriented towards hybrid vigor or "pure" (i.e. inbred) lines? The presence of inbreeding in natural populations may promote inbreeding depression. In this case, hybrid vigor is expected when crossing different inbred lines. However, if local adaptation has taken place in natural populations, hybrids might break down optimal allelic combinations for specific environments. Also, depending on the level of local genetic diversity, and how representative of this diversity the collection of the initial progenitors was, a breeding program could suffer from high rates of inbreeding and loss of allelic variation if the relationships between the breeding candidates were not considered when making selection decisions. Therefore, efforts to develop diversified germplasms for experimental evaluation of inbreeding effects and local adaptation may complement studies of natural populations, as well as promoting backup conservation strategies [START_REF] Barrento | Germplasm banking of the giant kelp: Our biological insurance in a changing environment[END_REF].

This study aims to investigate the spatial structure of the genetic and phenotypic diversities of the giant kelp, Macrocystis pyrifera (L.) C. Agardh. This species is under a strong and increasing exploitation pressure, mainly for alginate production and as a source of feed for abalone [START_REF] Buschmann | The status of kelp exploitation and marine agronomy, with emphasis on Macrocystis pyrifera, in Chile[END_REF]. Regulatory restrictions on kelp exploitation in many countries and the increasing demand for kelp biomass challenges the sustainable exploitation of natural populations, from which the large majority of the biomass is obtained. Biomass production through cultivation is an alternative that is being explored in several countries across its wide distribution range. In Chile, new legislation allows incentives for cultivation and repopulation of seaweeds, providing a positive environment for the installation of a kelp farming industry in the country. Pilot-production has demonstrated that 124 wet ton.ha -1 of M. pyrifera can be achieved using wild individuals to seed ropes for suspended systems [START_REF] Camus | Overview of 3 year precommercial seafarming of Macrocystis pyrifera along the Chilean coast[END_REF]. The development of M. pyrifera aquafarming is expected to emerge rapidly for several reasons: established procedures for cultivation in hatcheries [START_REF] Camus | Macrocystis pyrifera aquafarming: Production optimization of rope-seeded juvenile sporophytes[END_REF] and open ocean [START_REF] Camus | Overview of 3 year precommercial seafarming of Macrocystis pyrifera along the Chilean coast[END_REF] allow for the testing of the agronomic performance of a large array of genotypes and pilot scale production; technology used to convert biomass to bioethanol implemented at the pilot scale [START_REF] Wargacki | An engineered microbial platform for direct biofuel production from brown macroalgae[END_REF][START_REF] Camus | Scaling up bioethanol production from the farmed brown macroalga Macrocystis pyrifera in Chile, Biofuels, Bioprod[END_REF]; and identification of novel components for food and pharmaceutical uses that add value to the biomass production [START_REF] Wells | Algae as nutritional and functional food sources: revisiting our understanding[END_REF][START_REF] Ortiz | Functional and nutritional value of the Chilean seaweeds Codium fragile, Gracilaria chilensis and Macrocystis pyrifera[END_REF][START_REF] Leyton | Identification and efficient extraction method of phlorotannins from the brown seaweed Macrocystis pyrifera using an orthogonal experimental design[END_REF].

M. pyrifera is considered to be a highly plastic species [START_REF] Druehl | Morphological and growth responses of geographically isolated Macrocystis pyrifera populations when grown in a common environment[END_REF][START_REF] Demes | Phenotypic plasticity reconciles incongruous molecular and morphological taxonomies: The giant kelp, Macrocystis (laminariales, phaeophyceae), is a monospecific genus[END_REF], yet some morphological traits were considered to express a strong phylogenetic signal. Indeed, the spatial distribution of different morphotypes based on blade and holdfast shape along the coast was highly correlated with the presence of divergent clades of an ITS2-based phylogeny [START_REF] Astorga | Origin, diversification, and historical biogeography of the giant kelp genus Macrocystis: Evidences from Bayesian phylogenetic analysis[END_REF]. Using mitochondrial DNA, Macaya and Zuccarello [START_REF] Macaya | Genetic structure of the giant kelp Macrocystis pyrifera along the southeastern Pacific[END_REF] reported low genetic diversity across the South Eastern Pacific (SEP) but a concordance with the two major biogeographic discontinuities at 33°S, and 42°S, suggesting that environmental heterogeneity may be contributing to the distribution of the genetic diversity. Besides the relevance of this information, limited resolution of the molecular markers and the morphological survey restricts our understanding of the spatial patterns of phenotypic variation. The reduced genetic diversity and divergence among habitats or distant regions, and the high phenotypic plasticity were considered as strong arguments for a recent evolutionary history in the southern hemisphere where little or no adaptive divergence has occurred. Consequently, if natural selection had little or no impact on the species phenotypic diversity, it was considered that breeding and strain selection would be insufficient to modify traits and improve productivity under farming conditions. In this study, we challenged this view by developing a comparative study of genetic and morphological divergence across parts of the South American distribution range, with special emphasis on the region of Chiloé where environmental discontinuities are well known.

We quantified the genetic diversity and its spatial distribution in M. pyrifera across the SEP, and its association with morphological diversity. Secondly, we investigated the potential for modifying traits through controlled breeding by testing morphological, growth and chemical differentiation among crosses of M. pyrifera with different genetic backgrounds but cultivated in a common garden. Results are discussed in the context of seaweed domestication and sustainable production.

Materials and Methods

Morphological analyses

Adult sporophytes were collected at 16 locations along the Chilean coast (Table A1). At each site, between 20 and 30 mature individuals were collected along a transect of approximately 600 m by scuba diving, and transported immediately to the laboratory in boxes cooled with ice packs. Morphological analysis involved measuring the following characters of each thallus: number of stipes, total thallus length, total wet weight, number of blades, holdfast diameter and height. In addition, the following characters were measured for ten randomly selected blades per thallus: maximum blade length and width, blade angle with the stipe, maximum aerocyst length and width and substantiality (a measure of weight per projected blade area, expressed in g cm -2 ). To assess the variation of the set of morphometric characters and the correlations between them, a Principal Component Analysis (PCA) was performed. The level of structuring of the total sample based on morphological traits was assessed by a K-means clustering analysis that performs an iterative alternating fitting process of assigned individuals to a number of specified clusters (K = 2, 3 and 4) in order to maximize the morphological differentiation among groups. Finally, to evaluate the correspondence between morphology and genetic data, a discriminant analysis was performed assigning individuals to groups a priori defined from genetic clustering analyses, and the percentage of correct assignments was estimated as an indicator of the correspondence between morphological and genetic clustering (see below).

All multivariate analyses were performed with JMP 10 (North Carolina, USA).

Genetic analyses

A 3x2 cm piece of blade tissue was excised from each individual for 13 of the 16 collected populations (Dalcahue, Chaulinec and Meulin were not included, Table A1), washed with fresh water and immediately placed into a plastic bag with silica gel crystals for rapid dehydration. Total genomic DNA was isolated from finely ground tissue following [START_REF] Faugeron | Development and characterization of nine polymorphic microsatellite markers in the Chilean kelp Lessonia nigrescens[END_REF].

Nine microsatellite loci were selected from [START_REF] Alberto | Microsatellite markers for the giant kelp Macrocystis pyrifera[END_REF]: Mp-BC-4N; Mp-BC-13; Mp-BC-25; Mpy-7; Mpy-9; Mpy-11; Mpy-14; Mpy-17 and Mpy-19. PCR reactions were carried out according to [START_REF] Alberto | Microsatellite markers for the giant kelp Macrocystis pyrifera[END_REF] with minor modifications in annealing temperatures. PCR products were analyzed on an ABI3130xl Genetic Analyzer (Applied Biosystems, Foster City, California, USA) using 500 LIZ internal standard. Raw allele sizes were scored with GENEMARKER v1.95 and assigned to specific alleles using FLEXIBIN [START_REF] Amos | Automated binning of microsatellite alleles: Problems and solutions[END_REF].

Descriptive statistics for population genetic diversity, including number of alleles (Nall), observed (Ho) and expected (He) heterozygosities, estimators of inbreeding (F IS ) and pairwise population differentiation (F ST ) were calculated using GENETIX v 4.05.2 [START_REF] Belkhir | GENETIX 4.05, logiciel sous Windows TM pour la génétique des populations[END_REF].

Isolation by distance was evaluated by a Mantel test with 5000 permutations performed in GENETIX. The identification of genetic clusters was made using the Bayesian clustering approach implemented in STRUCTURE [START_REF] Pritchard | Inference of population structure using multilocus genotype data[END_REF]. The analysis considered possible admixture and correlated allele frequencies among populations as optional settings. The MCMC chain discarded the first 50000 iterations as burn-in, and kept only the subsequent 100000 iterations. The analysis was performed 10 times for each of the k-clusters (k = 1 to 12), and all these runs were integrated using STRUCTURE HARVESTER (available at http://taylor0.biology.ucla.edu/structureHarvester/). The uppermost likely number of cluster was defined following Evanno´s criteria Δk [START_REF] Evanno | Detecting the number of clusters of individuals using the software STRUCTURE: A simulation study[END_REF].

Germplasm collection and crossing experiment

Fertile sporophytes were collected from the sampled sporophytes at the 16 locations (Table A1). The germplasm collection was prepared following [START_REF] Barrento | Germplasm banking of the giant kelp: Our biological insurance in a changing environment[END_REF]. From the collection, three male and three female gametophytes from Puchilco (PUH) and Pargua (PAR) (PAR1♀, PAR20♀ and PUH6♀; PAR1♂, PAR20♂ and PUH6♂), were selected for their different genetic background (see Section 3.2.). These gametophytes were transferred from germplasm to new culture conditions to promote vegetative growth, following [START_REF] Lüning | Reproduction, growth and photosynthesis of gametophytes of Laminaria saccharina grown in blue and red light[END_REF][START_REF] Westermeier | A new approach to kelp mariculture in Chile: Production of free-floating sporophyte seedlings from gametophyte cultures of Lessonia trabeculata and Macrocystis pyrifera[END_REF].

Once sufficient gametophyte biomass was obtained, sexual fertility was induced following [START_REF] Westermeier | A new approach to kelp mariculture in Chile: Production of free-floating sporophyte seedlings from gametophyte cultures of Lessonia trabeculata and Macrocystis pyrifera[END_REF]. Both inbred and outbred crosses were performed. After 4-6 weeks, juvenile sporophytes were observed. F 1 sporophyte individuals (n= 30) of each cross were weighed, and morphological characters were measured. With the initial and final length and weight, the specific growth rate (SGR) was calculated as SGR = [(ln x 2ln x 1 )/(t 2 -t 1 )*100], where x 1 and x 2 are the measured trait at the beginning (t 1 ) and end (t 2 ) of the period.

After 15-20 weeks of cultivation under controlled conditions, between 100-300 individuals of each cross were transplanted to a 21-hectare outdoor floating cultivation system in southern Chile (Quenac; see [START_REF] Camus | Overview of 3 year precommercial seafarming of Macrocystis pyrifera along the Chilean coast[END_REF]). The sporophytes were attached to ropes, three individuals per meter, at 4 m depth and monitored for 4-7 months. Each month, three randomly selected individuals of each cross were collected to determine SGR. Also, the number of blades per sporophyte were counted. A fragment of blade tissue of each of these samples was cut, washed and dried in silica gel crystals for genetic analysis. When sporophytes reached maturity, indicated by the presence of sori, the individuals were removed and the reproductive tissue brought to laboratory to collect isolated female and male gametophytes for the germplasm collection. The rest of each of three individuals per cross was completely dried at 60°C for 24-48 h, milled and mixed to ensure homogenization for chemical analysis.

Chemical characterization

Carbohydrates (alginate, mannitol and glucans) were determined by first completing a 2-step enzymatic depolymerization. The first 24 h process used cellulases and alginatelyase to extract mannitol, convert all glucans to glucose and to solubilize all alginate.

Glucans and mannitol were determined via HPLC/IR. The second 24 h process used an oligoalginate-lyase to break all oligo-alginates into monomers. Ammonia was added in solution which spontaneously converts 4-deoxy-L-erythro-5-hexoseulose urinate (DEHU), an alginate monomer to 5-hydroxypyridine-2-carboxylic acid (5-HPA) which could be detected and quantified on HPLC/UV (for detailed protocols see [START_REF] Camus | Scaling up bioethanol production from the farmed brown macroalga Macrocystis pyrifera in Chile, Biofuels, Bioprod[END_REF]).

Fucoxanthin was quantified by HPLC, following [START_REF] Jaswir | Fucoxanthin extractions of brown seaweeds and analysis of their lipid fraction in methanol[END_REF]. Phloroglucinol was quantified using Folin-Ciocalteu method [START_REF] Koivikko | High-performance liquid chromatographic analysis of phlorotannins from the brown alga Fucus vesiculosus[END_REF] and Total Phenolic Content (TPC) was determined by the Folin-Ciocalteu method using Gallic acid as standard [START_REF] Heffernan | Phenolic content and antioxidant activity of fractions obtained from selected Irish macroalgae species (Laminaria digitata, Fucus serratus, Gracilaria gracilis and Codium fragile)[END_REF][START_REF] Auezova | Antioxidant activity of brown alga Saccharina bongardiana from Kamchatka (Pacific coast of Russia)[END_REF][START_REF] Cruces | Phlorotannin and antioxidant responses upon short-term exposure to UV radiation and elevated temperature in three south Pacific kelps[END_REF]. Finally, amino acids were quantified via acid digestion and derivatization followed by HPLC/UV detection [START_REF] Cohen | Compositional protein analysis using 6aminoquinolyl-N-hydroxysuccinimidyl carbamate, a novel derivatization reagent[END_REF][START_REF] Cohen | Synthesis of a fluoresecent derivatizing reagent, 6aminoquinolyl-N-hydroxysuccinimidyl carbamate, and its application for the analysis of hydrolysate amino acids via high performance liquid chromatography[END_REF].

Statistical analysis of strain selection

The growth rates and chemical concentrations of the nine strains were compared with a 1way ANOVA after assurance of normality and homoscedasticity. If significant differences were detected, a posteriori Tukey test was performed to identify the source of variation.

Growth rates based on the number of blades per individual at the hatchery stage were compared with a Kruskal-Wallis Test. Statistical analysis was performed in JMP 10.0.0.

Results

Microsatellite population structure

The nine microsatellite loci revealed 287 alleles in 373 genotyped individuals (5 -72 per locus; Table 1). The average number of alleles per population varied between 5.2 and 13.4 in Palqui and Pargua, respectively (Table 1). Heterozygosity for all populations, except Algarrobo, exceeds 0.5 with maximum values of 0.71 in Pargua. There was significant heterozygote deficiency (F IS < 0.01) in all populations but Los Choros and Puchilco, with significant values ranging from 0.048 in Pucatrihue to 0.250 in Antofagasta (Table 1).

Patterns of genetic structure

The STRUCTURE analysis indicated the existence of four distinct genetic clusters (Fig. 1):

Cluster 1 dominated by the Antofagasta population in northern Chile; cluster 2 comprised Punta Choros and Algarrobo, both in northern-central Chile; cluster 3 included southern Pacific and canal Chacao, from Chome to Pargua (FCO and PAR) populations, and cluster 4 included populations of the interior sea of Chiloé. Most the individuals had a high probability of belonging to their clusters, however a few (e.g. one to three individuals per cluster) showed an admixture with other clusters (Fig. 1). When assignment of individuals was restricted to only two clusters, the main genetic discontinuity separated northern (i.e. Algarrobo, Punta Choros and Antofagasta) from all southern populations. With three clusters, a new discontinuity appeared separating Chiloé populations of the interior sea from those of the wave exposed coast. Only Pargua, in the Chacao channel (separating Chiloé island from the continent) considered a protected coast site, was assigned to the open coast genetic cluster. Finally, the Antofagasta population appeared as unresolved, with mixed assignments of most its individuals. When defining a fourth cluster, Antofagasta appeared as a highly-differentiated population from all the other populations (Fig. 1). In summary, the analysis illustrated a clear pattern of spatial genetic differentiation within M. pyrifera populations along the SEP coast, with strong genetic discontinuities. No signature of isolation by distance was detected (Mantel test: R 2 = 0,024; p = 0,124).

Morphological variability

Three groups were identified in the PCA (Fig. 2). One was composed of individuals belonging to the northern populations only (Antofagasta, Los Choros and Algarrobo). The second included individuals collected on the exposed coast, south of the first group, and the Chacao channel (Chome, Mehuín, Pucatrihue, Faro Corona, Pargua) and one population from interior sea of Chiloé (Metri). The third group included the remaining individuals from the interior sea of Chiloé (Puchilco, Quenac, Palqui, Dalcahue, Chaulinec, Mehuín and Queilen).

The differences between the groups were explained by length, weight, disc diameter and number of blades, all of which had higher values in the interior sea of Chiloé. Sporophytes from the exposed coast had a distribution differentiated mainly by the number of stipes and blade width, with southern individuals having more stipes and thinner blades, closer in character to the pyrifera morphotype than northern individuals which were closer to the integrifolia morphotype. K-means clustering revealed strong differentiation between individuals from the interior sea of Chiloé, and the rest of the southern and northern populations when K = 2 (Fig. 3). For K = 3, two northern populations (Punta Choros and Algarrobo) were differentiated from the rest of the populations while Quenac (from interior Chiloé) formed a single populationcluster (Fig. 3). Finally, for K = 4, the clustering pattern was similar to the genetic clustering (Fig. 1), except that Quenac was still isolated in a different cluster and Antofagasta was not differentiated from the other populations on the exposed shores south of 33°S. Sporophytes found at Quenac had a particular morphology, with a pronounced conical holdfast, short but significantly wider laminae and longer aerocysts, which was distinct from other individuals of the interior sea.

Discriminant analysis of the morphological data using genetic clustering as a priori grouping revealed a high congruence of the spatial distribution of the morphological and genetic variability (Figure 4). The main difference was the population from Metri that was assigned to the same group as the northernmost population from Antofagasta. The results were consistent and revealed only 5.0%, 12.6% and 17.4% of misclassified individuals in K= 2, 3 and 4, respectively (Table 2). Independent of the level of structure, the correct assignment always exceeded 80%, revealing a strong correspondence between genetic and morphological data.

In a second discriminant analysis (Table 3), individuals were assigned based on environmental groups defined as the three biogeographical units recognized on the SEP coast [START_REF] Camus | Biogeografía marina de Chile continental[END_REF]: Peruvian province (18.4°-29°S), intermediate area (30° -41°S) and Magellan province (42° -56°S). The percentage of correct assignment decreased mainly for the northern genetic cluster (63%), with 31.5% and 5.6% incorrectly assigned to Pacific/Canal Chacao cluster and Chiloé cluster, respectively.

Crossing experiments

One female and one male gametophyte from each of three sporophytes with different genetic backgrounds, were selected from the established germplasm, based on contrasting morphological characteristics of the wild parental sporophytes, all were of the pyrifera morphotype (Table 4). PAR1 and PAR20 belong to the Southern Pacific-Chacao channel genetic cluster, and PUH6 to the Chiloé genetic cluster, all located in the interior sea (e.g.

same habitat but different genetic clusters).

Strong and significant differences between crosses were observed in growth rate using weight (g), length (cm) and number of blades per plant. Under hatchery conditions (Fig. 5), Bal 1 and Bal13 had the worst performance, whereas in the open sea culture (Fig. 6) they resumed their growth, and Bal 5 had the lowest growth rates under natural conditions. No significant differences were observed between the inbred and outbred crosses, neither in hatchery nor in open sea culture. Furthermore, holdfast morphology developed differently between crosses, with some extreme variability, i.e. from a well-developed structure (e.g. Bal 1, Fig. 7) to no holdfast (e.g. Bal 3, Fig. 7). In open culture, morphological differences were also observed between crosses (Fig. 8), but mainly in terms of total length and weight.

Chemical characterization

Chemical analyses were performed for all crosses, except Bal 3 that did not survive the culture conditions in open water. Carbohydrates, bioactive molecules and aminoacids exhibited strong variability and significant differences (Table A2) between crosses. These differences were on several occasions striking: alginate yield was over 3 times higher for Bal 14 than Bal 1, and mannitol was 7.7 times higher for Bal 14 than for Bal 1 (Figure 9A). This same situation was observed for two of the 3 bioactive compounds measured (phenols and phloroglucinol), Bal 14 had values more than 4 times higher for both compounds than Bal 9 (Figure 9B). In the case of aminoacids, 7 out of 16 showed significant differences between the crosses (Table A2). Six of the total number of aminoacids showed differences that did not vary significantly, but the other 11 aminoacids showed significant variation and Arginine and Leucine showed variations up to 2.5 times.

Discussion

Our analysis based on microsatellites markers and morphological data provides clear evidence of spatial structure within the distribution range of M. pyrifera along the SEP coast, with strong discontinuities in the distribution of both the genetic and the phenotypic diversities. Four major clusters were identified, which coincide with the geographic distribution of the populations (North, Central, South Pacific/Chacao channel, and Interior Sea of Chiloé). the lack of isolation by distance further supports the idea that, at the spatial scales considered in this study (from tens to hundreds of kilometers, and up to 2,600 km in total), the genetic diversity is structured into major clusters representing mainly regional groupings separated by discontinuities in the genetic identity of individuals. Some of these discontinuities are co-located with environmental breaks. For instance, a sharp discontinuity along the Chacao channel, in between the Island of Chiloé and the continent (35 km long and 4-6 km wide) separates the interior sea from the open coast, environments that differ in terms of wave exposure, salinity variation, water stratification and nutrient abundance. A second major discontinuity separates populations south of 33°S, characterized by strong but intermittent upwelling regimes, from populations north of 30°S dominated by weaker but more persistent over time upwelling [START_REF] Tapia | Latitudinal discontinuity in thermal conditions along the nearshore of Central-Northern Chile[END_REF]. These discontinuities correspond to previously described biogeographic boundaries (i.e. 30-33°S and 40-42°S) [START_REF] Camus | Biogeografía marina de Chile continental[END_REF][START_REF] Meneses | Patterns and breaking points in the distribution of benthic algae along the temperate Pacific coast of South America[END_REF] and are strongly associated with the phylogeographic discontinuities of a large number of invertebrates (see [START_REF] Haye | Phylogeographic structure in benthic marine invertebrates of the Southeastern Pacific Coast of Chile with differing dispersal potential[END_REF] and references therein) and seaweeds [START_REF] Guillemin | Phylogeography of seaweeds in the South East Pacific: Complex evolutionary processes along a latitudinal gradient[END_REF], which on occasions leads to speciation [START_REF] Tellier | Phylogeographic analyses of the 30°S south-east Pacific biogeograqphic transition zone establish the occurrence of a sharp genetic discontinuity in the kelp Lessonia nigrescens: Vicariance or parapatry?[END_REF]. Habitat heterogeneity plays an important role in kelp divergence by favoring adaptation to particular environmental conditions, as shown for the Lessonia species complex [START_REF] Tellier | Phylogeographic analyses of the 30°S south-east Pacific biogeograqphic transition zone establish the occurrence of a sharp genetic discontinuity in the kelp Lessonia nigrescens: Vicariance or parapatry?[END_REF][START_REF] Oppliger | Sex ratio variation in the Lessonia nigrescens complex (Laminariales, Phaeophyceae): effect of latitude, temperature and marginality[END_REF][START_REF] López-Cristoffanini | Tolerance to air exposure: a feature driving the latitudinal distribution of two sibling kelp species[END_REF][START_REF] Koch | Species separation within the Lessonia nigrescens complex (Phaeophyceae, Laminariales) is mirrored by ecophysiological traits[END_REF]. Phylogeographic analyses of M. pyrifera across the southern hemisphere have also revealed genetic discontinuities associated with these environmental frontiers [START_REF] Macaya | Genetic structure of the giant kelp Macrocystis pyrifera along the southeastern Pacific[END_REF] suggesting that the distribution of genetic diversity is strongly driven by the distribution of different habitats. The northernmost cluster, represented by a single sampled population (Antofagasta), does not appear to be isolated by any known environmental discontinuity. Analysis of other seaweed species along the SEP coast have indicated genetic discontinuities that do not coincide with biogeographic boundaries (e.g. Mazzaella laminarioides [START_REF] Montecinos | Species replacement along a linear coastal habitat: phylogeography and speciation in the red alga Mazzaella laminarioides along the South East Pacific[END_REF]), but with large interruptions in suitable habitat (e.g. long sandy beaches). Even though there is no such interruption of the rocky shore between Choros and Antofagasta, there is a total absence of M. pyrifera along a large section of coastline running approximately 600 km, south of Antofagasta [START_REF] Vásquez | Evaluación de la biomasa de praderas naturales y prospección de potenciales lugares de repoblamiento de algas pardas en la costa de las XV, I y II regiones[END_REF]. Such an interrupted distribution might be the cause of the significant differentiation of the Antofagasta population, as gene flow seems to occur over relatively short distances. Indeed, dispersal of this species is dominated by spore dispersal at scales of a few meters, leading to high inbreeding within and strong differentiation among populations [START_REF] Johansson | Looking into the black box: simulating the role of self-fertilization and mortality in the genetic structure of Macrocystis pyrifera[END_REF]. It is possible that the Algarrobo-Choros cluster, located within the 30-33°S biogeographic transition between the Peruvian Province and the Intermediate Area [START_REF] Camus | Biogeografía marina de Chile continental[END_REF], is poorly connected to the northern cluster because of both restrictions in dispersal due to the distances between populations and local adaptations caused by habitat divergence.

The strong concordance between morphological and genetic clustering further suggests environmental conditions are driving the evolutionary divergence between regions. Phenotypic plasticity has often been considered as an explanation for the diversity of phenotypic traits found along the coast. The morphological characters of the sporophytes considered here include those that used to be diagnostic for the distinction between M. integrifolia and M. pyrifera: holdfast shape, blade and aerocysts size. The observations of the integrifolia-morphotype along wave exposed rocky shores, and the pyrifera morphotype in the Interior Sea of Chiloé were considered plastic responses to the exposure to wave action (or the absence of it) [START_REF] Demes | Phenotypic plasticity reconciles incongruous molecular and morphological taxonomies: The giant kelp, Macrocystis (laminariales, phaeophyceae), is a monospecific genus[END_REF]. There is, however, evidence of genetic control of some traits, as demonstrated by the differential growth of juveniles of Macrocystis under variable nutrient concentrations within common garden experiments [START_REF] Kopczak | Variation in nitrogen physiology and growth among geographically isolated populations of the giant kelp, Macrocystis pyrifera (Phaeophyta)[END_REF]. Evidence of a phylogenetic signature of the morphological divergence between pyrifera and integrifolia types suggest that M. pyrifera is experiencing an incipient evolutionary divergence between the two morphotypes along southern hemisphere coastlines that can potentially be explained by the different environments that they inhabit [START_REF] Astorga | Origin, diversification, and historical biogeography of the giant kelp genus Macrocystis: Evidences from Bayesian phylogenetic analysis[END_REF]. However, gene flow is still occurring between both groups as indicated by laboratory results [START_REF] Westermeier | Sexual compatibility and hybrid formation between the giant kelp species Macrocystis pyrifera and M. integrifolia (Laminariales, Phaeophyceae) in Chile[END_REF], which are consistent with our results for a few admixed individuals in each cluster. Here, not only did we observe spatial clusters for morphological data, but when combining data sets in a discriminant analysis, the existence of these morphological clusters was well explained by their association with the genetic clusters. In other words, the species seems to be experiencing evolutionary divergence between different habitats. This reinforces the idea that the phenotypic diversity observed in M. pyrifera is an evolutionary response to environmental heterogeneity rather than pure phenotypic plasticity. The results of our limited crossing experiments, with as few as 9 male/female combinations, strongly reinforce this hypothesis. First, considerable variation was observed for all the analyzed traits, including shape, size, growth rate and chemical composition. Second, this limited sampling of natural diversity provided, after a single generation, evidence of strongly heritable variation, as each progeny was highly homogeneous in the common garden experiments (both in tanks and out-door), but very different from any other progeny. For instance, the observation of variation in the holdfast morphology, ranging from normal pyrifera type, to reduced structures, to total absence of a holdfast, is experimental proof of the strong genetic determinism of holdfast shape. Parental sporophytes all came from sheltered habitats, where selection for holdfast size may be weak and could allow individuals with small sized holdfasts to survive. Therefore, these populations may have retained some genetic variance for holdfast shape. This might not be expected in waveexposed populations where the drag forces eliminate individuals weakly attached to the substratum, and therefore tend to eliminate genetic variance for holdfast shape (i.e. purifying selection). These results may explain the phylogenetic signal of holdfast morphology previously described [START_REF] Astorga | Origin, diversification, and historical biogeography of the giant kelp genus Macrocystis: Evidences from Bayesian phylogenetic analysis[END_REF]. Such a hypothesis could be further tested by analyzing the variance of holdfast phenotypes in the progeny of sporophytes living in protected versus wave-exposed habitats. To conclude, holdfast shape may acquire some characteristics from the influence of the environment during early development [START_REF] Demes | Phenotypic plasticity reconciles incongruous molecular and morphological taxonomies: The giant kelp, Macrocystis (laminariales, phaeophyceae), is a monospecific genus[END_REF], but the genetic background of the different progeny is the main driver of variability in these common garden experiments. Similar conclusions can be drawn for the chemical composition, which also strongly suggests that sporophyte physiology is under genotypic control [START_REF] Kopczak | Variation in nitrogen physiology and growth among geographically isolated populations of the giant kelp, Macrocystis pyrifera (Phaeophyta)[END_REF], although seasonal variation is also known [START_REF] Camus | Overview of 3 year precommercial seafarming of Macrocystis pyrifera along the Chilean coast[END_REF]. Heritable variation of phenotypic diversity is one of the fundamental predictions of Darwin´s theory of evolution under natural selection. Altogether, our results strongly suggest that the diversity of phenotypic traits is under the strong influence of natural selection. Besides a recent evolutionary history of the species in the southern hemisphere [START_REF] Macaya | Genetic structure of the giant kelp Macrocystis pyrifera along the southeastern Pacific[END_REF], the amount and distribution of this heritable variation is likely the result of evolutionary divergence between the different habitats. Therefore, the usually recognized phenotypic plasticity of the giant kelp, as an explanation of its broad distribution, should be reconsidered and local adaptation should be experimentally tested among habitats.

The introduction of new varieties for seaweed cultivation is posing a number of biological challenges. For example, over reliance on genetically uniform breeds that, often have unstable performance and get discarded from the production lines after only a few years [START_REF] Zhang | Effect of domestication in the genetic diversity and structure of Saccharina japonica populations in China[END_REF]. This genetic homogeneity also increases vulnerability to environmental stress and pests, because of intensification [START_REF] Yong | Genetic identification and mass propagation of economically important seaweeds[END_REF]. However, these varieties have fixed certain economically important traits, questioning the influence of phenotypic plasticity in Laminariales. Many traits related to economic production and quality are quantitatively inherited, and determined by the combined interaction between genetic and environmental factors. Therefore, understanding the relationship between the genotype and the environment and their role in shaping phenotypes will accelerate our capacity to selectively breed and improve the agronomic performance of cultivated strains. Recent advances in QTL analyses of seaweed traits [START_REF] Zhang | Phylogeographic data revealed shallow genetic structure in the kelp Saccharina japonica (Laminariales, Phaeophyta)[END_REF] offer an alternative approach for demonstrating the role of the genetic background, and allow for a move towards the development of tools to assist selective breeding. Additionally, by suggesting that some processes of local adaptation are occurring in giant kelp populations along the SEP coast, our results should be relevant to the development of cultivars that fit into local/regional environments. Indeed, M. pyrifera as well as most other kelps that are being incipiently cultivated are still wild species that evolved genetic combinations that optimize the fitness of different genotypes in their local environment. Therefore, initial steps of selective breeding should assess unwanted consequences of breaking these optimal combinations and take into consideration the nature of the genetic resources and natural variation available in wild seaweed stocks, in order to achieve sustainable improvement of the agronomic performances of the cultivars in their native environment [START_REF] Valero | Perspectives on domestication research for sustainable seaweed aquaculture[END_REF]. In this context, wild-type genetic diversity needs to be tested under farming conditions and preserved and stored in germplasms [START_REF] Barrento | Germplasm banking of the giant kelp: Our biological insurance in a changing environment[END_REF] for subsequent breeding experiments.

In the current context of an increasing demand for seaweed biomass not only for hydrocolloids industry, but also for a much larger range of high value molecules for different industries, understanding and preserving the natural genetic diversity of the breedstock is a pre-requisite for developing efficient breeding strategies that will increase production through farming. Genotype and phenotype diversities within wild populations offer a large panel of interesting traits for the industry. We should also take advantage of the evolutionary history of the species, which has promoted genetic combinations optimized for the different habitats a species can occupy naturally. In this context, the high heritable variance for phenotypic diversity revealed by M. pyrifera represents a natural heritage, potentially highly valuable to the success of future breeding programs.

Acknowledgements

The authors acknowledge the support of Corfo-INNOVA through their program 09-CTEI-6866 and the Programa Basal from CONICYT (FB-0001). CC and SF also acknowledge the support of FONDECYT (1160930) and SF of Iniciativa Cientifica Milenio (ICM P10-033F).


P Loiseau

CHARACTERIZATION OF THE MICROSTRUCTURE OF ZIRCONOLITE-BASED GLASS-CERAMICS

Keywords: glass-ceramic, zirconolite, radionuclide conditioning, scanning electron microscopy

legislation in France has spurred research on enhanced separation and conditioning or transmutation of long-lived radionuclides from high level radioactive wastes (HLW). In this field, we have studied zirconolite-based glass-ceramics in which the crystalline phase (zirconolite: CaZrTi 2 O 7 ) aimed to preferentially incorporate minor actinides is embedded in a glassy calcium aluminosilicate matrix. At the laboratory scale, the crystallization of the parent glass is carried out thanks to a two-step thermal treatment: a nucleation stage followed by a growth stage. This paper presents the evolution of the crystallization, followed by scanning electron microscopy (SEM) and X-ray diffraction (XRD), with the temperature of the crystal growth thermal treatment, in the range 950° -1350°C.

INTRODUCTION

Research about new conditioning matrices for minor actinides (Np, Am, Cm), which would result from an enhanced separation of long-lived radionuclides from HLW, are currently investigated. The objective of this study is to develop zirconolite-based glass-ceramics that would preferentially immobilize minor actinides in the zirconolite crystalline phase. Thus a double containment is reached thanks to a homogeneous dispersion of the crystals in the glassy matrix. Zirconolite is a crystalline phase well known for its excellent containment capacity and long-term behavior (high chemical durability and self-radiation resistance) (1). The results presented in this paper deal with microstructural analysis of zirconolite-based glass-ceramics prepared by heat treatment of a parent glass. The influence of crystal growth thermal treatment temperature T c (ranging between 950° and 1350°C) on the microstructure of the materials was studied. The different crystalline phases formed after devitrification were characterized by their composition, size, shape, crystalline structure and their distribution in the residual glassy matrix.

SAMPLE PREPARATION AND EXPERIMENTAL METHODS

The glass composition studied is given in Table 1. It is based on the formulation established by the CEA-Marcoule [START_REF] Fillet | Environmental Issues and wastes Management Technologies in the Ceramic and nuclear Industries III[END_REF]3). All the samples were prepared using the following operating mode :

-melting at 1550°C in a platinum melting pot (10h) -pouring in water and grinding for glass homogenization before remelting at 1550° C (4h) -casting and quenching at room temperature on a metallic plate -thermal treatment at 810°C (slightly higher than the glass transformation temperature T g ≈ 775°C) for 2h: nucleation stage -thermal treatment at T c (950°-1350°C) for 2h: growth stage In order to study the intrinsic devitrification behavior of the parent glass, neither actinides nor simulants (lanthanides) were added to the composition. Results concerning the introduction of neodymium as actinide simulant are presented in other papers (4)(5)[START_REF] Loiseau | MRS Symp. Proceedings[END_REF]. Moreover these previous works have shown the occurrence of an opaque "crust" (a crystallized layer never exceeding 1 mm thickness) on the surface of the samples, while zirconolite crystals are homogeneously dispersed in the residual glass of the bulk for T c = 1000°-1200°C. Zirconolite probably forms in the bulk by homogeneous nucleation. The "crust" mainly consists of titanite (CaTiSiO 5 ) and anorthite (CaAl 2 Si 2 O 8 ) crystals that have nucleated heterogeneously on glass surface. SEM observations and elementary analysis were performed on cut and polished glass-ceramic sections using a JEOL 840A microscope coupled to an EDS PGT/IMIX analysis system. Powder XRD patterns of the bulk of glass-ceramics were recorded in θ/2θ mode using a Siemens D5000 instrument with CoKα wavelength (λ = 1.78897Å).

RESULTS AND DISCUSSION

SEM images and XRD patterns corresponding to the bulk of heat-treated samples are shown in Figures 1 and2 respectively. After nucleation (810°C 2h), the glass remains transparent; SEM and XRD reveal no microcrystallization in the bulk and on the surface of samples. This indicates that during this step, crystal growth rate is not significant.

For T c ranging from 950° to 1200°C a high density of star-shaped crystals (5-10 μm diameter) have nucleated and grown in the bulk. These crystals are homogeneously distributed and were identified by XRD: -For T c = 950°C, dendritic crystals have grown. XRD patterns indicate that they consist of a mixture of zirconolite and fluorite type phases. However only one kind of crystal morphology is observed by SEM. As there is no more fluorite type phase for T c higher than 950°C, it indicates that fluorite transforms into zirconolite at 950°C. These two phases probably differ only by cationic ordering [START_REF] Vance | [END_REF].

-For T c ranging from 1000° to 1200°C, zirconolite crystal morphology changes progressively. The dendritic microstructure which consists of main branches (corresponding to preferred growth directions) and secondary branches clearly becomes simplified for T c greater than or equal to 1100°C. Disappearance of these secondary branches can be associated with a progressive decrease of the residual supercooled liquid viscosity and consequently with a lowering of concentration gradients near the surface of the main branches (non-congruent crystallization). Interface stability is thus expected to increase with T c . At the same time, thickness of the main branches increases and a more open particle microstructure is observed. Moreover, XRD patterns evolution indicates that as T c increases, zirconolite lattice parameters progressively change (a splitting is observed for several XRD lines notably at 2θ = 35.5°, 42° and 59.5°). This evolution can be attributed to a progressive ordering of cations in the (Ca, Zr) planes of zirconolite (5).

From T c = 1250°C, a new crystalline phase identified as m-ZrO 2 (baddeleyite) forms at the expense of zirconolite which progressively disappears with increasing T c . These crystals appear as white elongated globular particles on backscattered SEM images. The strong contrast evolution between residual glass, zirconolite and baddeleyite is due to an increase of zirconium content in the corresponding phases. EDS analysis indicates that the baddeleyite crystals contain significant TiO 2 amount. A low CaO level is also detected. These results are in accordance with the existence of relatively wide solid solution domains between ZrO 2 on the one hand, and TiO 2 and CaO on the other hand (8,9). For T c = 1350°C, almost all zirconolite crystals have disappeared. They remain only as very thin needles decorating ZrO 2 particles. This shows that the dissolution limit of zirconolite is slightly higher than 1350°C. The fact that relatively large ZrO 2 crystals remain at this temperature indicates that this phase dictates the liquidus temperature of the system. It ban be noticed that the occurrence of baddeleyite at high temperature at the expense of zirconolite is in accordance with the phase diagram evolution published by Xu et al. (10) for the pseudo-binary ZrO 2 -(TiO 2 + CaTiO 3 ) system. The crystallized layer forming a "crust" on sample surface (not shown in Figure 1) is observed for all the samples prepared for T c lower than 1300°C. Its thickness progressively grows until 1150°-1200°C and then strongly drops as to disappear from T c = 1300°C.

CONCLUSION

The effect of crystal growth temperature T c on the microstructure and the structure of the crystalline phases formed in zirconolite-based glass-ceramics designed as durable waste forms for minor actinides immobilization was investigated. For T c ranging from 1000° to 1200°C, zirconolite is the only crystalline phase growing in the bulk. The cationic order in zirconolite crystals increases with T c and the dendritic microstructure observed for low T c progressively disappears. For T c higher than 1200°C, ZrO 2 crystals grow in the bulk at the expense of zirconolite and dictate the liquidus temperature of the system. Thus there is a 200°C safety margin for the preparation of glassceramics with zirconolite as the only crystalline phase in the bulk.

Figure 1 .

 1 Figure 1. Backscattered SEM images of the bulk of the glass-ceramics prepared at the indicated crystal growth temperatures T c (x 3000)

Figure 2 .

 2 Figure 2. XRD patterns of the nucleated glass and of the bulk of the glass-ceramics prepared at T c ranging from 950° to 1350°C ( fluorite type phase, zirconolite, baddeleyite)

Table 1 .

 1 Composition of the studied parent glass (weight %)

	Oxides	SiO 2	Al 2 O 3	ZrO 2	CaO	TiO 2	Na 2 O
	%	43.16	12.71	9.00	20.88	13.25	1.00


P Loiseau

D Caurant

N Baffier

C Fillet

NEODYMIUM PARTITIONING IN ZIRCONOLITE-BASED GLASS-CERAMICS DESIGNED FOR MINOR ACTINIDES IMMOBILIZATION

Keywords: glass-ceramic, zirconolite, minor actinide conditioning, neodymium partitioning

This study deals with glass-ceramic matrices designed for the conditionning of minor actinides, in which zirconolite crystals (CaZrTi 2 O 7 ) are homogeneously dispersed in a residual glassy matrix. Good immobilization performances require a high enrichment of actinides in the crystalline phase (double containment principle). Glass-ceramics are obtained by controlled devitrification of an aluminosilicate parent glass containing large amounts of TiO 2 and ZrO 2 . Neodymium was selected to simulate the trivalent minor actinides. Crystallization was performed at 1200°C for various Nd 2 O 3 contents (0 -10 wt. %). In all cases, zirconolite crystallization is obtained in the bulk of glass-ceramics. The evolution of Nd 3+ location between the crystals and the residual glass was followed by electron spin resonance and optical absorption. Both techniques demonstrate that neodymium is partly incorporated in zirconolite crystals. Moreover, total Nd 2 O 3 content in parent glass has a strong effect on Nd 3+ ions distribution.

INTRODUCTION

Current investigations are achieved on new matrices for the specific immobilization of minor actinides that would come from an enhanced reprocessing of nuclear spent fuel. Glass-ceramics containing highly durable crystals accommodating large amounts of actinides, such as zirconolite [START_REF] Ringwood | Radioactive waste forms for the future[END_REF], are good candidates. In this case, a high enrichment of actinides in the crystalline phase is required in order to get a double containment of radionuclides in crystals themselves embedded in a durable residual glass. Neodymium was selected to simulate minor trivalent actinides such as Am 3+ and Cm 3+ ions because of their similar ionic radii. For instance, in an eight-coordinated site, which is the coordination of the largest site (calcium) of zirconolite, the ionic radii of Nd 3+ and Am 3+ ions are respectively 1.109 and 1.09 Å [START_REF] Shannon | [END_REF]. Glass-ceramic samples were obtained by controlled devitrification at 1200°C of an aluminosilicate parent glass containing large amounts of TiO 2 and ZrO 2 and various Nd 2 O 3 contents (0 -10 wt. %). Zirconolite is the only phase that crystallizes in the bulk. But a crystallized layer, composed of titanite, anorthite and few baddeleyite crystals, forms at the surface (3). This layer whose thickness does not exceed 1 mm was separated from the bulk of glass-ceramics. The results, which are discussed here, focus on the characterization of bulk glassceramic by electron spin resonance (ESR) and optical absorption spectroscopies, particularly on the evolution of neodymium partitioning between the zirconolite crystals and the residual glass with total Nd 2 O 3 content.

EXPERIMENTAL

Parent glasses with various Nd 2 O 3 contents, ranging from 0 to 10 wt. %, were prepared on the basis of the following undoped glass composition (wt. %) (4,5):

SiO 2 (43.2), Al 2 O 3 (12.7), CaO (20.9), ZrO 2 (9.0), TiO 2 (13.2), Na 2 O (1.0) The compositions of Nd-doped glasses were calculated by keeping the relative proportions of all the other oxides constant. A two-step melting at 1550°C with an intermediate grinding stage was performed to obtain more homogeneous glasses. A 2 h nucleation stage at 810°C followed by a 2 h crystal growth stage at 1200°C led only to the crystallization of zirconolite in the bulk of all the hal-00177950, version 1 -9 Oct 2007 Author manuscript, published in "Atalante 2000 (les recherches scientifiques sur l'aval du cycle pour le 21ème siècle), Avignon :

France (2000)"

glass-ceramic samples, irrespective of their neodymium composition. Reference zirconolite ceramics having compositions of Ca

RESULTS AND DISCUSSION

The microstructure of zirconolite crystals formed in glass-ceramics bulk at 1200°C is quite insensitive to neodymium content. These crystals are lath-shaped: their length is around 10 µm and thickness is less than 1 µm [START_REF] Loiseau | Scientific Basis for Nuclear Waste Management XXIV[END_REF]6). Because of this submicronic thickness, EDX analyses of zirconolite crystals are imprecise, always including a contribution from residual glass. Table 1 reports three examples of EDX results concerning the 0, 6 and 10 wt. % Nd 2 O 3 glass-ceramic bulk. This confirms XRD results (7) which showed that neodymium incorporation inside zirconolite crystals increases without saturation effect when Nd 2 O 3 total wt. % ranges from 0 to 10 %. Moreover, these results indicate that neodymium is essentially incorporated in the calcium site of zirconolite structure with a charge compensation mainly ensured by a partial substitution of titanium by aluminum ions. Nevertheless, for the 10 wt % Nd 2 O 3 glass-ceramic, it seems that a part of neodymium (around 0.06 ions by formula unit) is not compensated by aluminum: these Nd 3+ ions are likely to equally occupy the calcium and zirconium sites of zirconolite structure, which maintains electroneutrality. On the other hand, it can be noticed that an important part of Nd 3+ ions remains unfortunately in the residual glass. This is mainly due to the low degree of crystallization of these glass-ceramics (less than 13 % in volume). Because of the difficulty to precisely evaluate the zirconolite composition and especially the degree of crystallization, indirect spectroscopic methods were used to estimate the molar ratio R of neodymium incorporated in the zirconolite crystals versus total neodymium content in the glassceramics (7). Figure 1 shows typical ESR and optical absorption spectra obtained for parent glass, glass-ceramic bulk and zirconolite ceramic. The differences observed in the ESR and absorption spectra of the three host matrices correspond to different local neodymium environments. The large inhomogeneous broadening (absence of resolution) of the optical transition lines in the glass absorption spectra (Figure 1Ba) is due to a distribution of neodymium environments in accordance with the amorphous nature of the matrix.

The optical absorption of Nd-doped zirconolite (Figure 1Bc) exhibits only four lines because the fifth stark level of 4 I 9/2 state is located at too high energy to be thermally populated at room temperature (Figure 1C). Glass-ceramic bulk spectra (ESR and optical absorption) can be decomposed into the contributions of residual glass and zirconolite crystals, as shown in Figures 1Ab and1Bb. To perform this decomposition, the residual glass signal was assimilated in both cases to the parent glass one (Figures 1Aa and1Ba), and was subtracted from the glass-ceramic bulk spectrum (Figures 1Ab and1Bb). The remaining spectra (finely dotted curves in Figures 1Ab and1Bb) are very close to the ones recorded for the Nd-doped zirconolite ceramic (Figures 1Ac and1Bc). This analogy indicates that neodymium environments are similar in zirconolite crystals prepared either by devitrification at 1200°C or by solid state reaction at 1460°C. R is first estimated from ESR glass-ceramics bulk spectra (Figure 1Bb), as follows. The double integration of Nd 3+ ESR signals in zirconolite crystals and in the whole of glass-ceramics (residual glass + crystals) are calculated; their ratio is equal to R. In the case of optical spectra, the determination of R is not as straightforward. Actually, the areas under zirconolite and residual glass optical absorption signals are not only proportional to Nd 3+ content, but also to oscillator strength of the 4 I 9/2 → 2 P 1/2 transition (transition probability). Comparison of optical absorption with ESR results allows the evaluation of the neodymium oscillator strengths ratio between the two phases (zirconolite / residual glass), giving access to a further optical estimation of R. Figure 2A shows the variation of R with Nd 2 O 3 total content. ESR and absorption results are in total agreement: R decreases with increasing Nd 2 O 3 total content. If the degree of crystallization in volume is assumed to be the same irrespective of total Nd 2 O 3 content, such a decrease in R would thus indicate that the neodymium concentration increases more slowly in the zirconolite crystals than in the residual glass with increasing total neodymium concentration (0 -10 wt. %). This would amount to a non-linear dependence of neodymium concentration in zirconolite (which is proportional to the number x of Nd 3+ ions per zirconolite unit formula) with total neodymium content in glass-ceramics. As x was not measured by EDX for all glass-ceramic compositions, it was calculated from R results. To perform this B A 4 I 9/2 2 P 1/2 C calculation, the degree of crystallization in volume was assumed to be constant and the x values determined by EDX for the 6 and 10 wt. % Nd 2 O 3 glass-ceramics were coupled to the corresponding R estimations (Figure 2B). Thus, it can be seen that neodymium concentration in zirconolite crystals increases slower when the Nd 2 O 3 total level is higher. At least one hypothesis can be put forward to explain this evolution. x may depend on a thermodynamic equilibrium governing neodymium incorporation between the crystalline phase (zirconolite) and the surrounding supercooled liquid (becoming residual glass after quenching). Then, x evolution could be explained by a variation of neodymium activity coefficients (at least in zirconolite, which is the most Ndconcentrated phase) with increasing Nd 2 O 3 total content (departure from a Henry's type law expected for diluted concentration, for example).

CONCLUSION

Zirconolite-based glass-ceramics were obtained at 1200°C. Various Nd 2 O 3 contents (0 -10 wt. %) were investigated as simulant of trivalent minor actinides. It was found that the ratio R (number of Nd 3+ ions incorporated in zirconolite crystals divided by Nd 3+ total content) decreases with increasing Nd 2 O 3 total content. This evolution would be mainly due to a non-linear dependence of x (number of Nd 3+ ions per zirconolite unit formula) versus Nd 2 O 3 total content. Nevertheless, x is a growing function of Nd 2 O 3 concentration in parent glass composition. Complementary EDX analyses are in progress to confirm the estimations of x made from the R results. Moreover, the use of other simulants (europium, gadolinium, thorium) is under study.

Figure 1 .

 1 Figure 1. Electron spin resonance (A) and optical absorption (B) spectra of (a) 2 wt. % Nd 2 O 3 parent glass, (b) glass-ceramic bulk (continuous-line curves), and (c) Ca 0.8 Nd 0.2 ZrTi 1.8 Al 0.2 O 7 zirconolite ceramic. (C) : schematic neodymium energy levels involved in 4 I 9/2 → 2 P 1/2 optical transitions for zirconolite.

Figure 2 .

 2 Figure 2. Evolution with Nd 2 O 3 total content of: (A) R estimated from ESR and optical absorption spectra of glass-ceramic bulk, (B) number x of Nd 3+ ions per zirconolite unit formula deduced from R.

  Oxford variable temperature accessory. Optical transmission experiments were carried out at room temperature on KBr pellets (≈ 200mg) containing 25 wt. % of Nd-doped material. Absorption spectra between 424 and 442 nm ( 4 I 9/2 → 2 P 1/2 Nd 3+ transition) were recorded on a Varian Cary 5E double beam spectrometer. This transition is interesting because the degeneracy of 2 P 1/2 state is not removed by the crystal field. At the most, the 4 I 9/2 ground state is decomposed by the crystal field into five Stark levels which are thermally populated following a Boltzman repartition. So, each type of neodymium site is characterized by a maximum of five lines whose positions depend on the crystal field.

	1-x Nd x ZrTi 2-x Al x O 7 (0 ≤ x ≤ 0.6) were prepared by solid state
	reaction at 1460°C. A PGT analyzer was used for energy dispersive X-ray (EDX) analysis
	(accelerating voltage of 15 kV, beam current of about 1.8 nA). Nd 3+ ion is a paramagnetic and
	optically active local probe; its electronic configuration and ground state are respectively 4f 3 and
	4 I 9/2 . Electron spin resonance (ESR) spectra were recorded on powdered samples (≈ 100mg).
	Typical measurements were made at 12 K with a microwave power of 20 mW, using a Bruker ESP
	300e spectrometer operating at X-band (ν ≈ 9.5 GHz) and equipped with a TE 102 rectangular cavity
	and an

Table 1 .

 1 Molar compositions determined by EDX for the phases formed at 1200°C in the bulk (residual glass and zirconolite) of glass-ceramics containing 0, 6 and 10 wt. % Nd 2 O 3 . Molar expected compositions of corresponding parent glasses are indicated in italics.Silicon detection in zirconolite analyses is indicative of residual glass contribution. Indeed, zirconolite is known not to easily incorporate silicon. As a consequence, the following rough estimations of zirconolite composition were deduced after removing residual glass contribution and excluding sodium (very low level): Ca 0.97 Zr 1.07 Ti 1.90 Al 0.06 O 7 for the undoped glass-ceramic, Ca 0.82 Nd 0.19 Zr 1.05 Ti 1.77 Al 0.17 O 7 for the 6 wt. % Nd 2 O 3 and Ca 0.76 Nd 0.26 Zr 1.04 Ti 1.74 Al 0.20 O 7.01 for the 10 wt. % Nd 2 O 3 glass-ceramics.

		SiO 2	Al 2 O 3	CaO	TiO 2	ZrO 2	Nd 2 O 3	Na 2 O
	0 wt. % Nd 2 O 3
	parent glass	48.84	8.48	25.33	11.28	4.97	0.00	1.10
	residual glass	54.49	9.43	25.63	7.17	2.23	0.00	1.05
	zirconolite	4.52	1.61	24.52	44.26	24.65	0.00	0.44
	6 wt. % Nd 2 O 3
	parent glass	48.23	8.37	25.01	11.14	4.90	1.27	1.08
	residual glass	53.68	9.10	25.00	7.39	2.38	1.21	1.24
	zirconolite	7.06	3.06	21.79	41.06	24.10	2.29	0.64
	10 wt. % Nd 2 O 3
	parent glass	47.77	8.29	24.77	11.04	4.86	2.20	1.07
	residual glass	52.61	8.89	24.72	7.69	2.56	2.09	1.44
	zirconolite	8.97	3.72	20.72	39.40	23.27	3.23	0.69