Caroline Gérard

Mathilde Valenchon

Nicolas Poulin

Odile Petit
email: odile.petit@cnrs.fr

Gérard Caroline

Valenchon Mathilde

Poulin Nicolas

Petit Odile

Thierry Bourgeois

Nans Burgarella

Rose Delacroix

Océane Liehrmann

Luc Martin

Virginie Nierat

Edouard Paumier

Louise Prévost

Julie Renard

Pierre Uhlrich

How does the expressiveness of leaders affect followership in domestic horses (Equus

Keywords: collective movement, consensus decisions, social status, personality, leadership, contagion mood

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

join the movement regardless of the condition. This positive and dynamic attitude can be perceived as an attractive feature by the followers. Finally, we evidenced collective arousal in followers initiated by the leader that could be essential for group cohesion. The present study is the first to investigate the expressiveness level in animals, and provides new insights into the emergence of leadership.

Introduction

The coordination and synchronization of individuals to maintain cohesion in social species requires collective decisions [START_REF] Conradt | Group decision-making in animals[END_REF]. Whatever the activity of the group, cohesion in social species is the only way to maintain the benefits of collective life, like reducing predation risk or increasing foraging success. Collective movements are an ideal context to investigate such collective decision-making, as group members have to agree about departure time and the direction in which they will move. The choice between different alternatives can be consistently made by one specific individual across events, such as the dominant male in gorillas (Gorilla gorilla beringei) (Watts, 2000) or the oldest female in African elephants (Loxodonta africana) [START_REF] Maechler | Matriarchs As Repositories of Social Knowledge in African Elephants[END_REF]. However, the decision can also be more consensual and involve a quorum or the whole group [START_REF] Conradt | Consensus decision making in animals[END_REF]. In this case, the decision is shared and the leadership is distributed between group members over contexts. This process may be the best means to manage any potential conflict of interest. Indeed, individuals from a given group differ in their age, sex, internal state and needs in many social species [START_REF] Petit | Decision-making processes: The case of collective movements[END_REF]. Therefore, although a shared decision cannot be totally optimal for all group members, it reflects an average of all individual preferences -via a quorum or the whole group -and may provide the best compromise by keeping the consensus cost low for each individual [START_REF] Conradt | Group decision-making in animals[END_REF].

During a consensual decision process, some individuals can have a stronger influence than others and are considered as leaders. This leadership is distributed among group members in many species [START_REF] Petit | Decision-making processes: The case of collective movements[END_REF]. For instance in groups of horses, every group member can initiate a collective movement [START_REF] Bourjade | Is Leadership a Reliable Concept in Animals? An Empirical Study in the Horse[END_REF][START_REF] Briard | Personality and Affinities Play a Key Role in the Organisation of Collective Movements in a Group of Domestic Horses[END_REF][START_REF] Briard | How stallions influence the dynamic of collective movements in two groups of domestic horses, from departure to arrival[END_REF], but some initiators appear to be more successful insofar that they are followed more quickly or display fewer failed initiations (in which no individuals follow the initiator). What makes a leader successfully trigger a collective movement? The location of individuals within the group when the movement starts is important. In groups of domestic geese (Anser domesticus) and cattle (Bos Taurus), individuals with a higher number of neighbors in close proximity are the most followed (Ramseyer et al., 2009a(Ramseyer et al., , 2009b)). However, location does not seem to be correlated to leadership success in horses [START_REF] Krueger | Movement initiation in groups of feral horses[END_REF]. In horses, variations in group activity, orientation and dispersion before departure play a key role in collective decision processes but are not correlated to leader success [START_REF] Bourjade | Decision-Making in Przewalski Horses ( Equus ferus przewalskii ) is Driven by the Ecological Contexts of Collective Movements[END_REF][START_REF] Briard | Personality and Affinities Play a Key Role in the Organisation of Collective Movements in a Group of Domestic Horses[END_REF]. Dominance status has been shown to be a critical factor in leader success, with high-ranking individuals producing a faster joining process in several species including horses [START_REF] Berger | Organizational systems and dominance in feral horses in the Grand Canyon[END_REF][START_REF] Feist | Behavior patterns and communication in feral horses[END_REF] and rhesus macaques (Macaca mulatta), (Sueur and Petit, 2008a).

However, more recent studies in horses did not confirm these results, showing a lack of correlation between dominance hierarchy and initiation success [START_REF] Bourjade | Is Leadership a Reliable Concept in Animals? An Empirical Study in the Horse[END_REF][START_REF] Briard | Personality and Affinities Play a Key Role in the Organisation of Collective Movements in a Group of Domestic Horses[END_REF]. The affiliative social network has been linked with inter-individual differences in initiation success in Tonkean macaques (Macaca tonkeana) (Sueur et al., 2009) and brown lemurs (Eulemur fulvus fulvus) [START_REF] Jacobs | Social Network Influences Decision Making During Collective Movements in Brown Lemurs (Eulemur fulvus fulvus)[END_REF]. However, social centrality is not correlated with initiation propensity in horses [START_REF] Krueger | Movement initiation in groups of feral horses[END_REF]. But inter-individual affinities do however explain the dynamic of the joining phase in terms of spatial organization, but not in terms of duration [START_REF] Briard | Personality and Affinities Play a Key Role in the Organisation of Collective Movements in a Group of Domestic Horses[END_REF]. Amongst individual factors, age and sex are important factors for the initiation of movements in several species. For instance, adult females initiate a majority of movements in European bison (Bison bonasus) [START_REF] Ramos | Collective decision making during group movements in European bison, Bison bonasus[END_REF], while male chacma baboons (Papio ursinus) initiate group movements more often than females [START_REF] Jacobs | Social Network Influences Decision Making During Collective Movements in Brown Lemurs (Eulemur fulvus fulvus)[END_REF]. Age or sex do not influence the initiation propensity or the joining latency in horses [START_REF] Bourjade | Is Leadership a Reliable Concept in Animals? An Empirical Study in the Horse[END_REF][START_REF] Briard | How stallions influence the dynamic of collective movements in two groups of domestic horses, from departure to arrival[END_REF][START_REF] Krueger | Movement initiation in groups of feral horses[END_REF]. However, as observed in baboons in high-risk contexts such as crop-raiding [START_REF] Schweitzer | Participant Profiling and Pattern of Crop-Foraging in Chacma Baboons (Papio hamadryas ursinus) in Zimbabwe: Why Does Investigating Age-Sex Classes Matter?[END_REF], personality has a strong influence with bold individuals initiating movements more frequently, in horses [START_REF] Briard | Personality and Affinities Play a Key Role in the Organisation of Collective Movements in a Group of Domestic Horses[END_REF]. Yet, these individuals are not more successful than others in the group. The relationship between energetic needs and leadership has also been explored, based on the hypothesis that an individual with higher energetic needs has a strong interest to initiate foraging movements more often and more successfully. This hypothesis has been confirmed in the plains zebra (Equus burchellii), where females are more likely to initiate movement when they are lactating [START_REF] Fischhoff | Social relationships and reproductive state influence leadership roles in movements of plains zebra, Equus burchellii[END_REF]. However, no consistent relationship has been found between energetic needs and leadership in horses [START_REF] Corbin | Collective behavior in horses: do physiological needs determine movement initiation?[END_REF].

At this point, we still need to pinpoint what makes an initiator more efficient than another in being followed by the group, thus making it a successful leader. In this study in the domestic horse, we propose to focus on the leader's attitude and its impact to the followers' recruitment. In several species, individuals that express signals such as glances, pauses or vocalizations are followed by more individuals than other initiators [START_REF] Meunier | How many for dinner? Recruitment and monitoring by glances in capuchins[END_REF]Sueur and Petit, 2008b). These signals can be voluntarily emitted by leaders and are supplemented by clues that the followers perceive in the behavior and/or posture of the leader. Despite their potential importance in the decision-making process of followers, these signals or clues have never been studied in horses. Inter-individual differences in the quantity or quality of signals or clues emitted by the leader could explain the different success rates observed in this species. This underlines the importance of determining which signals or clues are used by horses during an initiation. Furthermore, a combination of behaviors could enhance the accuracy of the message, regardless of whether it is sent to followers voluntarily or not (see « the backup signal hypothesis » [START_REF] Johnstone | Multiple Displays in Animal Communication:`Backup Signals' and `Multiple Messages[END_REF]). By recording these signals or clues, we expect to establish individual profiles and evaluate their stability over time and across contexts. Moreover, we will verify if some signals/profiles are linked to the personality of initiators. In a second step, we will test if a more expressive individual will be more followed than another initiator. The third step of this study is to analyze how social status of the leader influences the followership in terms of hierarchy and centrality. We hypothesize that a leader's expressiveness could differ according to its social status. However, we cannot predict the direction of that relationship. Indeed, both dominants and subordinates could be more expressive for opposite reasons, i.e. respectively, because of an over-confidence conferred by a greater social influence on others, or because of a need to compensate a weaker social position. Finally, we use the hypothesis of contagion mood [START_REF] De Marco | Collective arousal when reuniting after temporary separation in Tonkean macaques[END_REF] to examine the followership process. From the leader to the last follower, the level of expressiveness displayed by one individual could be transmitted to the next in the collective movement. This could explain the progressive and continuous joining process and could thus contribute to maintaining group cohesiveness. These hypotheses are tested by observing the collective movements of three stable groups of six females. The group departures are spontaneous or triggered by an experimental manipulation and continuously observed. The spontaneous context reflects collective movements that occur naturally while the experimentation simplifies the context and prevents any movement preparation. In the spontaneous condition, every group movement initiated by any group member is recorded. In the experimental condition, the group of horses is enclosed in a pen and one horse is removed from the group and shown where a food source is located. Next, the informed horse is returned to the pen and, then, the enclosure is opened.

With such a procedure, we decide upon the time of departure, the trajectory and the identity of the initiator. In both conditions, the leader's behavior at the initiation, the one of the followers when they joined the move and the joining latencies are systematically recorded and analyzed.

Materials and methods

Animals

Three groups of six female ponies (Equus ferus caballus) were observed for this study. Each group had been stabilized for at least six months before the beginning of the observation sessions and remained outdoors in pastures of at least one-hectare over the whole year (Group 1 and 2 in 2014 and Group 3 in 2015) at the Haut-Barr breeding farm in Saverne, France. Four individuals belonged to two different groups meaning that the analysis was based on fourteen individuals (mean age 7 ± 2 years). The hunger state of the individuals was presumably stable over time since they had ad libitum access to grass and hay.

Collective movements

Data used for this study concerned experimental and spontaneous initiations of movements of the three groups. All the videos were recorded by the same observer (MV).

Experimental condition

Before each initiation, the group was herded into a temporary pen (approx. 10 m x 10 m) and one individual was taken out of the group and informed about the location of a hidden food reward (i.e., 18 large-sized carrots placed out of the sight of the group, 45 to 50 m away from the pen entrance). Half of the carrots formed a stack, and the other half was distributed randomly around the main stack (up to 3m around) so that every individual could have access to food reward. In a second step, the informed animal was returned to the temporary pen. In a last step, the pen was opened and the informed horse could return to the food reward, thus initiating a collective movement. An initiation is defined as a movement of at least twenty steps away from the group without pausing and with the neck at or above the horizontal position [START_REF] Bourjade | Decision-Making in Przewalski Horses ( Equus ferus przewalskii ) is Driven by the Ecological Contexts of Collective Movements[END_REF][START_REF] Briard | Personality and Affinities Play a Key Role in the Organisation of Collective Movements in a Group of Domestic Horses[END_REF]. Similarly, a follower is defined as an individual which takes at least twenty steps away from the group in the same direction as the initiator. Every individual was tested at least four times in the role of the informed horse. If the informed horse did not initiate the movement, the test was considered to have failed.

Three cameras were placed along the way (outside the pasture and hidden by vegetation): the first at the departure point, the second at the location where the carrots have been placed, and the third halfway between these two locations. To become unpredictable for the horses, the food reward and the temporary pen could be placed randomly at different locations in the pasture, and the same location was never used twice on the same day or on two consecutive days. At the group level, we did not observe any increase of initiation rate or joining across the study (First vs. last trial: initiator's arrival latency: Mean1st trial±SE=112.2±38.3s, Meanlast trial±SE=72.67±26.8s, N=15, W=43.5, NS, Wilcoxon test; last follower's arrival latency/total number of followers: Mean1st trial±SE=36.7±11s, Meanlast trial±SE=14.5±3.7s, N=15, W=30.5, NS, Wilcoxon test; number of followers: Mean1st trial±SE=4.5±0.3, Meanlast trial±SE=4.9±0.1, not enough variability for statistical analysis). Failed initiations occurred even during the last tests and the number of tests at the individual level was too small to encourage any habituation process.

Spontaneous condition

Each group was filmed during continuous and daily sessions of approximately six hours.

The observation periods ran for nine consecutive days for a total recording period of 50 hours minimum per group. The criteria used to define an initiation of a collective movement and the joining processes in these experiments are identical to those described in the previous section.

Data collected

The data were all scored by the same person (CG) who was unaware of the working assumption at the scoring period. For every collective movement, video analysis was used to calculate the joining latencies between the initiation and the departure of each follower. Only movements involving at least one follower were considered. The success of a leader was estimated by calculating the total joining latency (i.e. the joining latency of the last follower divided by the number of followers).

The focal animal sampling method [START_REF] Altmann | Observational study of behavior: sampling methods[END_REF] was used to record the behavior of leaders from departure to arrival for every initiation. The movement was discarded from the analysis when the initiator was not visible from the departure to the arrival or was no longer visible when the first follower departed. Four behaviors were considered to be perceptible signs for followers of the expressiveness of the leader. They were based on behaviors that are classically observed in literature about recruitment (Cebus capucinus: glances, pauses and speed [START_REF] Meunier | How many for dinner? Recruitment and monitoring by glances in capuchins[END_REF]; Cygnus Cygnus: shaking head [START_REF] Black | Preflight signalling in swans: a mechanism for group cohesion and flock formation[END_REF]). The occurrence and proportion of the following behaviors were collected from the initiation until the first individual followed the initiator or until the initiator arrived at its destination: gait (presence of trot and/or gallop), raised head position (i.e., the time during which the initiator held its head raised), head shaking (number of events), pauses (duration) and glances (number of events). The two last behaviors occurred in only three initiations and were therefore excluded from the analysis. An expressiveness score was calculated according to the presence or absence of the three remaining behaviors. The score calculation was decided to avoid an overestimation of one behavior over another. The scale ran from zero (none of the three behaviors was emitted) to three (the three behaviors were emitted).

The behavior of other group members, including both followers and non-followers, was recorded using the scan sampling method [START_REF] Altmann | Observational study of behavior: sampling methods[END_REF]. A first scan was performed at the initiation step to assess whether group members could see the leader, as there are hedges in the pastures that can hide the initiator. A second scan was performed at the departure of every follower to record its joining rank as well as its head position, gait and head shaking. In this way, the expressiveness score was calculated for every follower.

If one of the followers was not visible when it departed, the movement was discarded from the analysis. In total, 42 experimental and 74 spontaneous movements were used for the analysis.

Assessing dominance rank and centrality

Agonistic interactions between individuals were continuously recorded during the observation sessions in the spontaneous condition. Unidirectional interactions corresponded to approaches, threats or aggressions which are responded to by clear avoidance alone. SOCPROG 2.5. was then applied to obtain a David's score [START_REF] David | Ranking from unbalanced paired-comparison data[END_REF] for each individual.

The centrality index, or eigenvector, was based on proximity (Whitehead, 2009). During the observation sessions, inter-individual distances were collected every fifteen minutes with the scan sampling method [START_REF] Altmann | Observational study of behavior: sampling methods[END_REF] and 200 scans were collected per group. Two individuals were considered close when the inter-individual distance was less than one meter. Indeed, this distance enables the individuals to interact without moving. The eigenvector was obtained using SOCPROG 2.5 and represents the total number of connections an individual has, including the connections between its partners. The higher this index is, the more central the individual is within the group.

Assessing personality

Personality tests were performed in spring 2014 on 12 individuals according to a simplified standardized protocol (for further details see [START_REF] Lansade | Development of personality tests to use in the field, stable over time and across situations, and linked to horses' show jumping performance[END_REF]. Finally, we obtained 11 indices of fearfulness and 10 indices of curiosity. The missing data for 4 individuals are explained by the fact that one horse was reluctant to move away from the group to be tested, interfered with the curiosity test of another one, and that these tests could not practically be repeated in 2015. The aim was to evaluate the reactivity of an individual to a novel or sudden stimulus in order to characterize one personality dimension: fearfulness. For every individual, three tests were successively performed in a precise order: unfamiliar object, unfamiliar surface and suddenness. An additional test was performed to evaluate curiosity.

During the first test, the handler led the horse around an unfamiliar object (Fig 1a) with a slack leading rein. Three zones were defined around the object: the first corresponded to a distance of up to two meters, the second was between two and four meters and the last was four meters or more. The number of the zones that the horse crossed at every quarter turn was recorded. The addition of these numbers for the entire turn provided a score that was higher if the horse was frightened. The number of glances towards the object was also recorded.

In the unfamiliar surface test, a bucket with appetent food was presented to the horse and then placed at the center of a 2m x 2m black and orange plastic sheet (Fig 1b). Then the leading rein was left slack so the horse was free to move. The latency until the horse put one foot on the sheet to access the bucket was recorded as well as the time taken to eat the food.

The suddenness test consisted of rapidly opening and closing a large umbrella in front of the horse from a five-meter distance, then from a three-meter distance. The escape distance in each repetition was measured and a mean was calculated.

Additionally, a curiosity test (not included in [START_REF] Lansade | Development of personality tests to use in the field, stable over time and across situations, and linked to horses' show jumping performance[END_REF] was performed with the same object as the one used for the unfamiliar object test. The advantage of performing this test after the previous ones is that the object was no longer unknown. This made it possible to measure curiosity reactions while excluding the fearfulness dimension. The approach latency and the number of physical contacts with the object (sniffing, nibbling or nuzzling) over a 180 s period were recorded.

Statistical analysis

All statistics were computed using the R 3.3.2 statistical environment (R Core Team 2016).

Individual profiles of expressiveness

This analysis is the only analysis taking into account the identity of the leader. The individuals belonging to two different groups across years were considered as two different individuals in the analysis to assess an eventual change of expressiveness between the two years and social environment. A hierarchical cluster analysis with Ward's method was performed to classify initiations according to the initiator's expressiveness level. Time proportion spent raising head, time proportion spent galloping or trotting and shaking head occurrence variables were included (agnes function of Cluster package [START_REF] Maechler | Matriarchs As Repositories of Social Knowledge in African Elephants[END_REF]. The catdes function of FactoMineR package [START_REF] Lê | FactoMineR: an R package for multivariate analysis[END_REF], including a khi-2 test, was used to test the independence of qualitative variables (group and condition) and excluded the possibility of a group-effect (overrepresentation of a particular group in one category). The same function was then used to describe the obtained clusters and investigated which variables (both qualitative and quantitative) were characterizing the clusters.

Expressiveness effect on followership

The total joining latency was analyzed with a linear model using the expressiveness score, time proportion spent raising head, time proportion spent galloping or trotting, shaking head occurrence and condition as fixed effects (lme function of nlme package [START_REF] Pinheiro | Linear and Nonlinear Mixed Effects Models[END_REF]). Individuals were nested in groups as a random effect, initiations then become anonymous and avoid group bias. Normality and homoscedasticity of residuals were visually inspected (plot.lme and qqplot.lme functions of nlme package [START_REF] Pinheiro | Linear and Nonlinear Mixed Effects Models[END_REF]). The joining latency was converted into logarithm to fit with a normal distribution. All variables were screened for excessive multicollinearity (VIF >10 due to the low number of variables) (vif function of car package [START_REF] Fox | An R Companion to Applied Regression[END_REF]). This adjusted model included level 2 interactions with an LRT test approach (lmer function of lme4 package [START_REF] Bates | Package "lme4[END_REF]).

A first model solely included the initiations in which the initiator was visible to at least the first follower (N= 112; 38 in the experimental condition and 74 in the spontaneous condition).

A second model solely included the initiations from the previous selection in which the expressiveness score of the initiator was above zero (i.e. 1, 2 or 3, N=40; 21 in the experimental condition and 19 in the spontaneous condition). The aim of this second model was to assess the expressiveness effect only when it was present.

Social status effect on followership

The same method was used to analyze total joining latency, using David's score and eigenvector as fixed effects and individuals nested in groups as random effects. To avoid experimental bias, only the spontaneous condition was considered. In a first step, we only included initiations in which the initiator was visible by at least the first follower (N=74). A second model was then built that solely included the initiations from the previous selection in which the expressiveness score of the initiator was above zero (i.e. 1, 2 or 3, N=19). Finally, a third model was built for initiations in which the expressiveness score of the initiator was null (N=55). As previously, normality and homoscedasticity of residuals in every model were visually inspected (plot.lme and qqplot.lme functions of nlme package [START_REF] Pinheiro | Linear and Nonlinear Mixed Effects Models[END_REF]). The joining latency was converted into logarithm to fit with a normal distribution. All variables were screened for excessive multicollinearity (VIF >10 due to the low number of variables) (vif function of car package [START_REF] Fox | An R Companion to Applied Regression[END_REF]). This adjusted model included level 2 interactions with an LRT test approach (lmer function of lme4 package [START_REF] Bates | Package "lme4[END_REF]).

Contagion mood in followers

The expressiveness score of every follower (Sn) was examined in successive generalized linear mixed models. The expressiveness scores of the previous individuals in the movement (from S0 to Sn-1) were used as fixed effects and the individuals nested in a group and the condition as random effects. The score variable was transformed by dividing it by 3 (the maximum number of expressive behaviors), thus permitting the use of a binomial family error distribution in the model. All variables were screened for excessive multicollinearity (VIF >10 because of the low number of variables) using the vif function of car package [START_REF] Fox | An R Companion to Applied Regression[END_REF]. This model was adjusted using an LRT test approach (glmer function of lme4 package [START_REF] Bates | Package "lme4[END_REF]). We selected the initiations involving the whole group from all the initiations in which the initiator was visible by at least the first follower (N=46; 36 in experimental conditions and 10 in spontaneous conditions).

Results

Individual profiles of expressiveness 1) Experimental condition

The hierarchical cluster analysis extracted two categories: initiations with a low or a high level of expressiveness by the leader. The distribution is balanced, with 20 initiations in the first category (low expressiveness) and 22 in the second (high expressiveness). The leader is expressive in 52.40% of initiations. The means of the three behaviors significantly differ between low and high expressiveness category (time proportion spent raising head, low: 0.00±0.00% vs. high expressiveness category: 74.70±5.33%, P<0.001; time proportion spent galloping or trotting, low: 2.50±2.44% vs. high expressiveness category: 56.06±7.23%, P<0.001; shaking head occurrence, low: 0.00±0.00 per hour vs. high expressiveness: 0.36±0.10 per hour, P<0.05). Furthermore, some individuals are seen in only one category whereas others can be found in either category, depending on the initiations. Finally, the mean of the curiosity score is significantly higher in the high expressiveness category (Fig 2) (P<0.05), suggesting that the expressiveness is associated with a higher curiosity trait.

2) Spontaneous condition

The signs of expressiveness are less frequent in the spontaneous condition than in the experimental condition, occurring in only 17.60% of the initiations. However, the same categories, i.e., a low (61 initiations) or a high (13 initiations) level of expressiveness, were extracted for the spontaneous condition. Here again, the individuals are either found in one specific category or alternate between the two, but their distribution differs from the experimental condition.

Like in the experimental condition, the means of the three behaviors significantly differ between low and high expressiveness category (time proportion spent raising head, low: 3.28±2.28% vs. high expressiveness category: 61.15±25.60%, P<0.0001; time proportion spent galloping or trotting, low: 0.00±0.00% vs. high expressiveness category: 18.57±9.84%, P<0.001; shaking head occurrence, low: 0.00±0.00 per hour vs. high expressiveness: 0.62±0.12 per hour, P<0.001). Furthermore, the means of the curiosity scores are not significantly different between the two categories. Nevertheless, the eigenvector (Fig 3

Effect of expressiveness on followership

After the analysis of all the initiation data collected, the best predictor model includes the condition variable, indicating that joining latency is significantly longer in the spontaneous condition than in the experimental one (Table 1) (Student test, Df=1, t=3.43, P < 0.001). The best predictor model for initiations in which the expressiveness score of the leader is not null is the one that only included the score variable, which is negatively and significantly related to joining latency (Table 1) (Student test, Df=1, t=-2.23, P < 0.05). It suggests that the more expressive the leader is, the faster the followers will join the movement.

Social status effect on followership

With all the initiations included, the best predictor model for joining latency is the null model, suggesting that neither dominance nor centrality status has a clear effect. We obtain the same result for the initiations in which there is no sign of expressiveness (i.e. score null) and those in which we can observe an expressive leader (i.e. score equal to 1, 2 or 3). Thus, whatever the level of expressiveness expressed by the leader, its dominance and centrality status are not clearly related to its success.

Contagion mood in followers

In each of the successive models, the best predictor model only includes the score of the individual immediately preceding each follower in the movement (Table 2). Thus, the expressiveness of the first follower (S1) is positively and significantly related to the expressiveness of the leader (S0) (Wald test, z=2.01, P<0.05). Similarly, the expressiveness of the second follower (S2) is positively and significantly related to the expressiveness of the first follower (S1) (Wald test, z=3.34, P<0.001). The score of the third follower (S3) is also positively and significantly related to the score of the second joiner (S2) (Wald test, z=4.80, P<0.001).

Finally, the expressiveness of the fourth follower (S4) is positively and significantly related to the expressiveness of the third follower (S3) (Wald test, z=3.50, P<0.001). This rule does not apply for the last follower, as its expressiveness score (S5) is positively and significantly related to that of the third follower (S3) (Wald test, z=2.92, P<0.05).

Discussion

Individual profiles of expressiveness

These results show a high degree of variability in the expressiveness of leaders across initiations. This is illustrated by the presence of two different categories. The first (low expressiveness category) includes the initiations during which few or no signs of expressiveness were observed in the leader's behavior, and the second (high expressiveness category) concerns the initiations in which the leader is highly expressive. Between these two categories, the means of the three behaviors are significantly different, indicating that these variables are efficiently discriminant, without any one of them being more representative than another. These clusters enable us to validate the relevance of the behaviors we selected and the expressiveness score we built.

This study fails to distinguish stable individual profiles of expressiveness. Nevertheless, the results show a higher propensity to initiate being more expressive in the experimental condition (54.2% of the initiations in the high expressiveness category) than in the spontaneous condition (only 17.6%). This result is not surprising since the experimental procedure aims to increase the motivation of initiators with a highly appetent reward in order to provoke direct and standardized initiations. However, it must be specified that, even in the spontaneous condition, we obtained large variations in the score of expressiveness between the different initiations. Furthermore, in the experimental condition, the curiosity score of the expressive leaders (high expressiveness category) is higher than that found in leaders displaying no sign of expressiveness. It indicates that curious individuals seem to be more prone to initiate with a higher level of expressiveness. This relationship is not observed in the spontaneous condition, highlighting the importance of context in the relationships between personality and leadership. Changes from the context that animals routinely encounter could enhance the motivation of curious individuals to initiate, and thus explain why this result is only obtained in the experimental condition. Although personality has never been shown to be a necessary factor for individual success, this link between personality and leadership has previously been revealed in horses, where bold individuals were seen to initiate more often than others [START_REF] Briard | Personality and Affinities Play a Key Role in the Organisation of Collective Movements in a Group of Domestic Horses[END_REF]. The same results were obtained in Olive baboons (Papio hamadryas ursinus), where males initiated raids on crops more often than females. All of these could be explained by a propensity to be active in unusual and novel contexts [START_REF] Schweitzer | Participant Profiling and Pattern of Crop-Foraging in Chacma Baboons (Papio hamadryas ursinus) in Zimbabwe: Why Does Investigating Age-Sex Classes Matter?[END_REF].

In the spontaneous condition, the dominance and centrality indexes of the leader are lower in the high expressiveness category. This indicates that the subordinate and/or peripheral individuals seem to be more prone to be highly expressive when initiating movements. It may suggest that these individuals compensate their status by their level of expressiveness. Indeed, a high hierarchical position has been shown to facilitate leader success (in gorillas (Gorilla gorilla) (Watts, 2000), wolves (Canis lupus) [START_REF] Mech | The wolf: the ecology and behavior of an endangered species[END_REF], and mongooses (Helogale undulata rufula ) [START_REF] Anne | Dwarf mongoose and hornbill mutualism in the Taru desert, Kenya[END_REF]). Nevertheless, our results are based on a restricted number of individuals. The findings would benefit being supported by future additional studies. Finally, if the level of leader's expressiveness has a real effect on the followers, individuals with a low social status could compensate for their relatively weak position and influence group members by being more expressive than the dominant and central initiators and consequently obtain similar levels of success. It has been shown that horses might have a representation of their social position in their group [START_REF] Krueger | Horse sense: social status of horses (Equus caballus) affects their likelihood of copying other horses' behavior[END_REF], but this phenomenon of compensation also raises the question of a horse's ability to be aware of how its own behavior influences others. However, caution should be taken when considering the intentionality of such a behavior, as operant conditioning would suffice to explain the emergence of expressiveness. Indeed, the expressive behavior could have been associated with a positive reaction from another individual (i.e. success), thus leading to positive reinforcement [START_REF] Bouton | Learning and behavior: a contemporary synthesis[END_REF]. This could therefore be a case of social conditioning, insofar that individuals adjust their behavior according to the reactions of their congeners from a very early age [START_REF] Ducoing | Withholding information in semifree-ranging Tonkean macaques (Macaca tonkeana)[END_REF].

The effect of expressiveness on followership

When all initiations are analyzed, the only significant effect on the joining latency is the condition. The joining process is faster in the experimental condition than in the spontaneous condition. Here again, the result can be explained by the motivation increase caused by the reward. If we consider the higher frequency of expressive initiations in this condition, we can conclude that this increase in motivation affects both the leaders and the followers through increased expressive behaviors and joining speed, respectively. Indeed, as a grazing species, healthy horses are known to be constantly motivated by highly palatable food (McGreevy and Mc Lean, 2010), and carrots are very appetizing. As a consequence, motivation for food might be an important driver for all the group members to move more in the experimental condition than in the spontaneous condition. However, this increase in motivation can also be explained by all the parameters that are generally favorable for both initiating and following in the experimental condition, such as the spatial proximity, the good visibility on each other's behavior, or the fact that trajectories are direct. Nevertheless, no causality link can be established between leader expressiveness and joining speed at this stage.

When only the initiations in which the leader showed at least one of the expressiveness behaviors are included, the expressiveness score appears to be negatively and significantly related to the joining latency. It therefore seems that the level of expressiveness shown by the leader increases its success in terms of joining speed, but only if the expressiveness score is above zero. Indeed, if the leader does not express any sign, the joining latency varies from one extreme to another and is probably dependent on other parameters. However, if the leader expresses these signs, they seem to become a decisive factor for the decision-making process of group members.

Effect of social status on followership

Similar to the conclusion drawn about previous results in horses [START_REF] Bourjade | Is Leadership a Reliable Concept in Animals? An Empirical Study in the Horse[END_REF][START_REF] Briard | Personality and Affinities Play a Key Role in the Organisation of Collective Movements in a Group of Domestic Horses[END_REF], we do not find any significant relation between the dominance status or centrality of a leader and its success in terms of joining speed. We expected this study to explain the absence of a consistent link between dominance status and leadership in previous studies in horses [START_REF] Bourjade | Is Leadership a Reliable Concept in Animals? An Empirical Study in the Horse[END_REF][START_REF] Briard | Personality and Affinities Play a Key Role in the Organisation of Collective Movements in a Group of Domestic Horses[END_REF] by an additional factor that hid this effect, but no evidence was found that expressiveness was responsible for this absence of a link in the population studied.

Contagion mood in followers

This study shows that the score of expressiveness of the preceding individual in the movement is positively and significantly related to the next follower. This result suggests a chain reaction that spreads expressiveness from the leader through to the fourth follower and then to the final one. The result for the fifth follower is necessarily biased owing to its position in the movement. Its individual decision process is probably strongly influenced by the risk of social isolation if it does not follow. This contagion mood could be one of the modalities explaining the effect of leader expressiveness on joining speed. Every follower may transmit a kind of impulse to the next individual, enhancing its motivation. This type of phenomenon has already been observed in animals, and particularly in a positive context following the reunion of individuals that had been experimentally separated. These events, named collective arousal, demonstrated a contagion mood based on non-verbal communication, imitation and synchronization processes between individuals [START_REF] De Marco | Collective arousal when reuniting after temporary separation in Tonkean macaques[END_REF].

Finally, like in mimetic processes [START_REF] Camazine | Self-organization in biological systems[END_REF], only the first follower needs to be influenced by the leader expressiveness for this influence to be transmitted through every follower's behavior. This mechanism could therefore efficiently maintain the impulse of the leader and finally enhance the social cohesion of the group [START_REF] De Marco | Collective arousal when reuniting after temporary separation in Tonkean macaques[END_REF] and its coordination [START_REF] Bono | Charisma, positive emotions and mood contagion[END_REF]. We think that the contagion mood via a mimetic process is a very interesting process that merits further investigation, as this mechanism may allow group cohesion for groups of all sizes.

Conclusion and perspectives

The present study demonstrates that horses can be more or less expressive when initiating a collective movement, and that this expressiveness can help the leader to be followed. This positive and dynamic attitude can be perceived as an attractive feature by the followers and could be close to the leader's charisma as found in humans. Interestingly, the expressiveness observed in our study was mainly shown by subordinate individuals and more often by socially peripheral group members, suggesting that they may increase their level of expressiveness in order to be followed. This potential compensation of social status through behavior by subordinate and peripheral individuals may support the theory of a form of emotional intelligence in horses i.e., the capacity to recognize and influence the emotions of congeners. Indeed, in humans, regardless of variations in his/her own emotional state, the leader can use his/her intelligence to maintain a positive attitude whatever the situations [START_REF] Bono | Personality and Transformational and Transactional Leadership: A Meta-Analysis[END_REF]. Such emotional intelligence could enable individuals to have voluntary control over their behavior and moderate their expression depending on the individuals around them (Walter and Bruch, 2009). Finally, this attractiveness of the leader through its positive and dynamic attitude, as well as this possible kind of social compensation, shapes a leadership pattern that recalls the charismatic leadership model developed in the human literature (Walter and Bruch, 2009).

To conclude, our study provides the first analysis of expressiveness in animals and yields some encouraging avenues for future research. Furthermore, we believe that the concept of charisma could be applied to a non-human species and is a very promising opportunity for future research about leadership origins.

Finally

  , two Principal Component Analyses with R 3.1.3 (R Core Team, 2015) including all the results of the different tests provided a score for each individual for the fearfulness dimension (75.80% on Dimension 1) and the curiosity dimension (72.56% on Dimension 1, see Online Resource 1 for figure).

  ) and David's score means (Fig 4) are significantly higher in the low expressiveness category (eigenvector: P<0.05; David's Score: P<0.001) suggesting that the expressiveness is associated with low centrality and subordinate status.

Villot for help with the experimental task. A USIAS grant awarded to O.P. funded the overall project.

Ethical Note

All the experiments undertaken in this study complied with ethical standards of French research practices. The authorization number is AL/01/10/07/11 for the Regional Ethical Comity for Animal Experimentation (CREMEAS). Neither procedure adversely affected the horses in the short term or for the overall period of the study. Sueur, C., 2011. Group decision-making in chacma baboons: leadership, order andcommunication during movement. BMC Ecol. 11, 26. Sueur, C., Petit, O., 2008a

Captions

Table 1: The best predictor models for the joining latency

The first line represents the best predictor model when all the initiations were included. The second line represents the best predictor model for the initiations in which the expressiveness score of the leader is equal to zero.

Table 2: The best predictor models for the expressiveness score of the followers

The only variable that is significantly related to the expressiveness score of an individual is the score of the direct previous individual in the movement. The last joiner is not included in this rule.


Kevin Crouvisier-Urion

Fernanda Regina Da

Silva Farias

Sorawit Arunatat

Donnchadh Griffin

Massimiliano Gerometta

Jeancarlo Rocca-Smith

Guy Weber

Nicolas Sok

Thomas Karbowiak
email: thomas.karbowiak@agrosupdijon.fr

Kevin Crouvisier Urion

Fernanda Da

Jean-Carlo Rocca Smith

Functionalization of chitosan with lignin to produce active materials by waste valorization

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

Introduction

Lignin is an abundant natural amorphous polymer and a cheap by-product of paper production, cellulosic industries and ethanol bio-refineries, [1][2][3][4] which annually produce millions of tons of this polymer. 5 Lignin is the second most abundant component in wood after cellulose (18-35 %wt of the dry matter). [START_REF] Pettersen | The chemistry of solid wood[END_REF] Not only is it one of the main components of wood, but also of fruits and vegetables, representing 20 %wt of the worldwide biomass. [START_REF] Rosillo-Calle | The biomass assessment Handbook: Bioenergy for a sustainable environment[END_REF] It is difficult to characterize the exact chemical composition of lignin because of the extraction process from the biomass, into which non-native chemical species can be introduced. 8 Nevertheless, a general empirical molecular formula for the lignin monomer can be determined as C 9 H (10-n) O 2 (OCH 3 ) n , with n being the mean ratio of the methoxy groups on the aromatic rings of the lignin monomer (Figure 1). Based on the value of n, the three phenyl propene lignin monomers can be divided into the following units: the phydrophenyl unit (n = 0; H unit), the guaicyl unit (n = 1; G unit) and the syringyl unit (n = 2; S unit). 4,9 These units are commonly referred to as coumaric, ferulic, and sinapic acids when present as free monomers, respectively. The macromolecular structure of lignin depends on the ratio and random polymerization of the monomers, according to their botanical origin. 10,11 The resulting lignin is an important structural polymer in the plant cell wall, providing antimicrobial resistance, low permeability, and high mechanical resistance. 9,12 Another interesting property of lignin is its anti-oxidant activity due to the hydroxyl groups on the aromatic ring (Ar-OH, MeO-Ar-OH, (MeO) 2 -Ar-OH). 13 Currently, lignin is mostly used as a low-cost fuel. However, recent studies described the use of this by-product as a valueadding commodity in polymer production. One of the strategies adopted for lignin valorization is the incorporation into polymer matrices, 14,15 using blending, 16 solvent casting, 13,17,18 layer-by-layer 19,20 or spin coating. 21 These Please do not adjust margins Please do not adjust margins methods have already been applied in the production of conventional polymers, such as polypropylenes (flame retardants), 3 polystyrenes (filler applications), 10 or carbon fibres and polyacrylamides (material reinforcements).

22,23

Furthermore, more sustainable matrices such as starch, 24 soy protein, 25 and chitosan 13,17,18 can also be used for application as films. Indeed, chitosan is a potential candidate for the replacement of conventional plastics due to its film-forming capacity (giving transparent and resistant films) and high oxygen barrier property. 13,26,27 This is also a waste product from the marine industry that can be valorized. Its structure, derived from chitin deacetylation, is shown in Figure 2. The incorporation of lignin is targeted to confer antioxidant activity 13 or UV-barrier property 28 to packaging materials in order to protect sensitive products, such as foods or cosmetics, against oxidation. However, chitosan and lignin are non-thermodynamically compatible polymers since chitosan is hydrophilic and soluble in aqueous phase, while lignin is hydrophobic and soluble in organic phase. Because of this poor compatibility, the physical incorporation of lignin into chitosan matrix leads to a heterogeneous system in which lignin aggregates are distributed within the polymer network.

13,17

This even occurs at a very low lignin percentage (> 2.5 %wt).

13

Physical treatment, such as high pressure processing, can improve the lignin dispersion but also induces migration of lignin residues.

18

The objective of this study was to investigate the use of using chemical grafting to improve the compatibility of chitosan and lignin and to assess whether grafting preserves anti-oxidant activity. In the present work, grafting was carried out with lignin, but also with the different lignin monomers, in order to identify the origin of lignin's anti-oxidant activity and the effect of grafting using a systematic comprehensive approach.

Experimental Material

Chitosan with a deacetylation degree of 90 %, was supplied by France Chitin (France). Its weight-average molecular weight was 274 kDa (±32), and its number-average molecular weight was 163 kDa (±9). 29 Lignin used for this study, provided by Granit SA (Swiss), was extracted from sugar cane (Saccharum munja) by an alkaline process. This type of lignin is usually referred to as "Sarkanda". It has a G/S unit ratio of 1.04 and a low content of free phenolic monomers with around 0.5 mg.g -1 .

The three acid phenols corresponding to the three lignin monomers (Figure 1) used in this study were coumaric acid (98 % purity), trans-ferulic acid (99 % purity) and sinapic acid (98 % purity). Acetic acid (99 % purity), ethanol (99.8 % purity), Nhydroxysuccinimide (NHS; 98 % purity) and N-(3dimethylaminopropryl)-N'-ethylcarbodiimide hydrochloride (EDC; 98 % purity) were purchased from Merck (Germany).

Grafting of lignin monomers and lignin polymer onto chitosan

The synthesis reaction was adapted from the protocol described by Woranuch et al. (2013), 30 and a molar ratio of chitosan monomer to lignin monomer close to one was used. First, a chitosan solution (1.2 % w/v) was prepared by dispersing 0.6 g of chitosan in an aqueous solution composed of 0.5 mL of acetic acid and 49.5 mL of distilled water at 25 °C for 12 h. To obtain an activated ester of each lignin monomer, 0.648 g of ferulic acid, 0.548 g of coumaric acid, or 0.748 g of sinapic acid, were mixed with NHS (0.384 g) and EDC (0.518 g) in 10 mL of ethanol for 30 min. The same protocol was used to obtain activated lignin polymer, using 0.6 g of lignin.

Afterwards, this activated ester solution of lignin monomer or lignin polymer (10 mL) was added into the chitosan solution (50 mL), and the final solution was stirred for 12 h at 60 °C (pH ≈ 6). The mixture was then dialysed (using 3500 Da molecular weight cut off for the lignin monomers and 100 000 Da molecular weight cut off for the lignin) with distilled water as buffer. To ensure elimination of free acids and by-products of the reactions, dialysis was repeated 10 times within 5 days. Self-standing films (of around 40 µm thickness) were obtained by casting and solvent evaporation of the dialysed solution, at 25 °C and 50 % relative humidity.

Characterization of the grafting reaction onto chitosan

Elemental analysis was carried out using a Thermo Electron Flash EA 1112 Series CHNS/O analyser. Around 1 mg of sample was pyrolysed with excess oxygen at 960 °C, and three replicates were analysed. UV-Vis absorbance spectra were recorded at a wavelength range of 200-800 nm (Safas mc1 UV-Vis spectrophotometer). After grafting and dialysis, the casting solution was analysed, using acetic acid solution (1 % v/v) as blank, and lignin monomer (1 % m/v) in acetic acid solution (1 % v/v) as control. Fourier Transform InfraRed (FTIR) absorbance spectra of the starting materials (lignin monomers, lignin and chitosan) and of the grafted films were recorded at 25 °C at a spectral range from 400 to 4000 cm -1 using a Bruker Equinox 55 spectrophotometer equipped with a global source, DTGS detector and KBr splitter. Absorbance spectra were recorded in transmission mode with a resolution of 2 cm -1 and an average of 100 scans per analysis, using a KBr pellet (200 mg) as reference. For sample preparation, KBr pellets were pressed after mixing 199 mg of KBr and 1 mg of solid material.

Anti-oxidant activity

A DPPH• (α-diphenyl-β-picrylhydrazyl) test was used to measure the Radical Scavenging Activity (RSA) of films, . Lignin cannot be tested with this method since there is an overlapping of the absorbance of DPPH•. For the films (with or without grafting), the samples were prepared by stirring 10 cm 2 of each film in 10 mL of an ethanol DPPH• solution at 50 mg.L -1

. The final concentration of the DPPH• solution was thus fixed at 50 mg.L -1 both for the powder and the film samples.

The kinetics of the reaction were followed at 25 °C by the disappearance of the DPPH• free radical using absorbance measurement at λ = 515 nm with a UV-visible spectrophotometer (Biochrom WPA lightwave II). The DPPH• solution (2 mL) was sampled and returned to the original solution after each absorbance reading. Samples were maintained in closed vials covered by aluminium foil to protect them from light and stirred (200 rpm), until the reaction equilibrium was reached.

The reduction in radical scavenging activity, RSA(t), in %, was expressed as:

Results and discussion

Grafting of lignin monomers onto chitosan

Lignin monomers were grafted onto chitosan by ester activation through a carbodiimide-mediated coupling reaction in acetic acid solution (1 % v/v) with EDC and NHS, using a molar ratio of lignin/chitosan monomer close to one. By using this method, the coupling agents transformed the acid function of the ferulic acid (RCOOH) into a reactive ester (RCOOR). Amide linkage was created by nucleophilic substitution of the ferulic activated ester on the reactive primary amine function of the chitosan, 30,33 as shown in Scheme 1.

In order to validate the grafting reaction of the lignin monomers onto chitosan, UV-Vis spectroscopy was first performed on the solution obtained after the grafting reaction and dialysis, and just before casting (Figure 3). In the range of 250-800 nm, chitosan in solution did not exhibit any absorbance peak. However, after the grafting reaction with lignin monomers, absorbance peaks were noticed. Chitosan solutions grafted with coumaric acid, ferulic acid, and sinapic acid, displayed characteristic absorbance peaks with maxima at 309, 316, and 317 nm, respectively. These values were in Please do not adjust margins

Please do not adjust margins the same range as those obtained for the corresponding monomers in solution: 340, 320, and 322 nm for coumaric, ferulic, and sinapic acid, respectively. 34,35 However, a slight shift was observed in the maximum absorbance values. This has already been identified as a consequence of the grafting reaction. 36,37 UV-Vis spectroscopy measurements, performed on the solution obtained after grafting and dialysis, thus clearly indicated successful grafting.

To go further in the analysis of the grafting, FTIR absorbance spectra were also determined, first on lignin monomers and chitosan (Figure 4) and then on grafted films obtained after casting (Figure 5). Firstly, coumaric acid showed characteristic peaks (Figure 4a (ω C-H of the pyranose ring). 30,[51][52][53][54][55] Finally, when analysing the FTIR absorbance spectra obtained from the chitosan grafted films (Figure 5), it was noteworthy that, for all three films grafted with lignin monomers, shifts occurred from 1603 cm -1

( N-H of the amine I groups) down to around 1560 cm -1 ( N-H of the amine II groups). This was completely in accordance with the grafting reaction (Scheme 1), because the grafting took place on the amine group of the deacetylated chitosan monomer units, which therefore change the local environment of the corresponding nitrogen atom. Furthermore, the characteristic peaks of each monomer also appeared on the corresponding film spectra, especially peaks attributing to stretching vibrations of the aromatic ring (around 1520 and 1462 cm -1 ),  C-O of the hydroxyl groups for ferulic acid (1277 cm -1

) and β C-H of the aromatic ring for sinapic acid (1114 cm -1

). The presence of these new absorbance peaks in the final composite polymer was thus additional evidence that lignin monomers were successfully grafted onto chitosan. Following spectroscopic analysis, that proved the successful 1: Elemental composition of starting materials (calculated from their empirical formula for pure lignin monomers) and of grafted films (n=3), given their respective standard deviation. C/N ratio and grafed yield (expressed as molar %) are also reported. Please do not adjust margins Please do not adjust margins grafting, the yield of the reaction was quantified for each lignin monomer, based on the elemental analysis of the starting materials and grafted films (Table 1). An increase of the carbon/nitrogen ratio in the grafted polymers was observed compared with the initial chitosan. Considering that nitrogen in the grafted films only derived from chitosan and that additional carbon content in the final polymers was brought by chemical grafting, this allowed the calculation of the reaction yields. The resulting reaction yields were close to 13.8, 13.2 and 12 % for coumaric, ferulic, and sinapic acids, respectively (Table 1). The yield of grafting for each lignin monomer was not very high, although grafting based on carbodiimide ester activation was supposed to be highly efficient in grafting phenolic compounds onto chitosan. 56 However, the grafting reaction may have been greatly affected by the reaction conditions such as chitosan deacetylation degree and molecular weight, initial molar ratio of chitosan/phenolic acids, pH, temperature, and time. 30,57,58 Furthermore, in this study, ethanol was used as a green solvent for the grafting reaction, but ethanol can also inhibit the reaction by acting as a reactant.

59

Grafting of lignin onto chitosan polymer

To further examine the grafting process, the reaction was performed not only with lignin monomers, but also with lignin polymers and evaluated whether this waste product can be valorized without a prior monomer extraction step. UV-Vis, FTIR, and elemental analyses were used as described before to validate the grafting and to quantify the yield of reaction.

In acetic acid (1 % v/v), lignin showed a characteristic absorbance peak at 280 nm. 60 After reacting with activated lignin, chitosan also exhibited a peak at 280 nm (Figure 6), which indicated successful grafting. FTIR analysis revealed the presence of lignin on the grafted films (Figure 7). In the FTIR absorbance spectrum of lignin (Figure 7b, brown line), the characteristic peaks of "Sarkanda" lignin could be identified as: 3414 cm -1 ( O-H of the hydroxyl groups), 2922 cm -1 ( C-H of the aromatic methoxyl groups), 2851 cm -1 ( C-H of the methyl-and methylene groups), 1701 cm -1 ( C=O of the unconjugated carbonyls), 1600 cm -1 and 1515 cm -1 ( C=C of the aromatic ring and  C=O of the conjugated carbonyl), 1462 cm -1 ( as C-H ), 1425 cm -1 ( C-H of the aromatic ring), 1218 cm -1 ( C=O ,  C-C and  C-O ), 1120 cm -1 ( C-H of the syringyl units), and 1031 cm -1 ( C-H of the aromatic ring,  C-O of the hydroxyl-and ether groups). [61][62][63][64][65][66][67] With regard to the FTIR spectrum of lignin grafted onto chitosan (Figure 7a, purple line), it is worthy to note that there was a shift from 1603 cm -1

( N-H of amine I) down to 1566 cm -1 ( N-H of amine II). This shift was related to amine I of deacetylated chitosan monomers, which highlighted a change in the local environment of the reacting amine function (as was previously observed with the lignin monomers). Additional characteristic peaks of lignin also appeared in the spectrum, especially peaks attributed to the aromatic ring vibrations and C-H vibrations of the methoxy groups at 1518 cm -1 and 2846 cm -1

, respectively. The presence of these new absorbance peaks in the final composite polymer unambiguously showed that the lignin had successfully been grafted onto chitosan. Regarding the monomers, the grafting yield was calculated following the same procedure as described above, which is based on elemental analysis. However, the sulphur content of lignin was used for normalization, because nitrogen was detected both in chitosan and lignin powder (Table 1). The resulting grafting yield was around 9.7 %. It is noteworthy that this yield of grafting was slightly lower than the one obtained for each individual monomer. This could be explained by the steric hindrance of this macromolecule, which could have limited the lignin grafting. Once the grafting method was proved to be effective, and the yield of the reaction was quantified, it was then important to Figure 6: UV-VIS absorbance spectra obtained on the film forming solution after the grafting reaction (followed by dialysis) of lignin onto chitosan (CS: chitosan -LG: lignin). Chitosan and lignin spectra in solution are also displayed for comparison purpose. The arrows point out maximum characteristic absorbance peak. Please do not adjust margins Please do not adjust margins evaluate the anti-oxidant properties of the resulting films to consider potential applications of this method to produce active packaging materials.

Anti-oxidant activity of lignin and lignin/chitosan grafted films compared to the initial free lignin monomers

The RSA of starting and final grafted materials was evaluated using the DPPH method. Figure 8 displays the anti-oxidant properties of acid phenol lignin monomers. Coumaric acid exhibited the lowest RSA (5 % at equilibrium), while ferulic acid displayed an intermediate value (35 %). Among the three lignin monomers, sinapic acid showed a significantly higher RSA, leading to a 70 % decrease of the DPPH free radicals at equilibrium. This difference in the anti-oxidant activity of lignin monomers could easily be explained by their chemical structure. Lignin monomers are composed of two hydroxyl groups with different degrees of methoxy substitution (Figure 1). Indeed, coumaric acid has no methoxy groups, whereas ferulic acid is ortho-methoxy substituted, and sinapic acid is di-orthomethoxy substituted. As reported by Brand-Williams et al. (1995), the ortho-methoxy substitution favours the stabilization of the aryloxyl radical by electron donation, which therefore increases the radical scavenging activity of the molecule. 31,68 These results, therefore, highlight that the respective ratio of lignin monomers can strongly influence the global anti-oxidant activity of the lignin polymer. In order to obtain stronger anti-radical activity, lignins could be selected based on their constitutive monomers (high sinapic acid and a low coumaric acid content). The same trend was observed when the monomers were grafted onto chitosan. The chitosan films grafted with coumaric acid displayed almost no anti-oxidant activity (slightly higher than the chitosan films alone), and the one grafted with ferulic acid showed intermediate anti-oxidant activity. Furthermore, sinapic acid grafted films remained the most efficient to scavenge DPPH• free radicals (Figure 9). At equilibrium, the films grafted with coumaric acid led to a decrease of around 9 % of free DPPH• radical, compared with around 50 % for ferulic acid and 90 % for sinapic acid grafted films. Furthermore, although there was a low grafting yield of lignin onto chitosan, a decrease of around 50 % of the free radical scavenging activity was observed when the lignin was grafted onto chitosan. Based on RSA values obtained for ferulic and sinapic acid at equilibrium (50 and 90 %, respectively) and on the ferulic/sinapic monomer ratio of "Sarkanda" lignin (about 1.04), a theoretical RSA value of around 70 % at equilibrium can be expected for grafted lignin films. This clearly highlights that the maximum radical scavenging efficiency of grafted lignin films has not been reached. This could be explained by the non-availability of some hydroxyl functions due to steric hindrance or the involvement of reactive groups in the intramolecular bindings of the lignin polymer. This hypothesis was supported by the reaction kinetics of lignin with DPPH•. Indeed, the equilibrium was not fully reached for the films grafted with lignin compared with those grafted with the monomers. However, the diffusion of DPPH• molecules in the film matrix may have been slowed down, and this could explain that the reaction kinetics were decreased compared with lignin monomers grafted films, in which the hydroxyl groups are more easily accessible. Moreover, as grafting occurred with chitosan, there was obviously no migration of active molecules in the solution. Nevertheless, despite a grafting yield of around 10 % and taking into account that reactive groups would not be available, chitosan films grafted with lignin exhibited improved capacity to scavenge free radicals and could be an interesting way to valorize these two industrial waste products and to use them as active materials.

Conclusions

This study shows that grafting of lignin monomers onto chitosan is feasible, using an intermediate reaction with NHS and EDC as coupling agents, in ethanol as a green solvent.

Grafting was detected by UV-Vis absorption, as well as by FTIR spectroscopy, with a shift of the peak attributed to the amine I of chitosan, where the grafting occurred, and with the concomitant appearance of absorbance peaks attributed to the aromatic rings from lignin monomers. In addition, based on carbon, nitrogen and, sulphur content, reaction yields of around 13 and 10 % were obtained for lignin monomers and Please do not adjust margins Please do not adjust margins lignin, respectively. Even if a low reaction yield added antioxidant activity to the grafted films, it could be worthwhile to improve the grafting reaction yield. The anti-oxidant activity of lignin monomers and grafted films was also investigated in this work. Significant differences in the anti-oxidant activity of lignin monomers were noticeable, with a higher radical scavenging activity of sinapic acid due to di-ortho-methoxy substitution. In contrast, coumaric acid displayed no anti-oxidant activity, and ferulic acid had an intermediate activity. Additionally, the anti-oxidant activity of lignin monomers was preserved after grafting onto chitosan, and the respective order of the anti-oxidant activity remained the same. After grafting, the radical scavenging activity occurred without the migration of active substance in the liquid phase in contact. Furthermore, these results also show that the composition and the ratio of lignin monomers influence the anti-oxidant activity of lignin. Therefore, lignin should be selected based on its constitutive monomers. It will be interesting to determine the anti-oxidant activity of lignin and lignin grafted onto chitosan, which has been extracted from several lignin taxonomies in order to confirm the hypothesis that the higher the syringyl unit concentration is, the higher the anti-oxidant activity becomes. It is also possible to improve the chemical extraction of lignin, and to preferentially select a fraction containing a higher content of sinapic acid and a low content of coumaric acid. This could lead to the identification of the best lignin source, and extraction processing, for the production of active materials, in order to protect sensitive products from oxidation, such as foods or cosmetics, for extending their shelflife. This first proof of concept sets up the basis to further improve and tune the anti-oxidant activity of the final product.

Figure 1 :

 1 Figure 1: General condensed formula of lignin monomer, with detailed chemical structure of the three monolignols corresponding to the degree of methyl-substitution on the aromatic ring. The global value of n depends on the respective proportion of these three sub-units in the lignin: H (p-hydroxyphenyl), G (guaiacyl) and S (syringyl).

Figure 2 : 3

 23 Figure 2: Structure of the chitosan polymer (at pH > pKa amino group) displaying both acetylated and deacetylated monomer units.

  where A sample (t) is the absorbance measured for the DPPH• solution containing the sample at time t, and A blank (t) is the absorbance of the DPPH• solution at the same time. This correction takes into account the kinetics of DPPH• autodegradation. Thus, RSA(t) values represent the anti-oxidant capacity of the sample. Kinetics measurements were done in triplicate, at least.

Scheme 1 :

 1 Scheme 1: Chemical pathway for ferulic acid grafting onto chitosan using ester activation of the lignin monomer. The coupling agents (NHS and EDC) transformed the acid function of the ferulic acid (RCOOH) into a reactive ester (RCOOR). The intermediate activated ester reacts on the amine I of the chitosan deacetylated unit, by nucleophilic substitution. Coumaric and sinapic acids, as well as lignin, were also grafted following the same reaction.

1 (

 1 , blue line) at 3377 cm -1 ( O-H of the hydroxyl groups), 3072-2495 cm -1 ( O-H of the carboxylic acid groups and  C-H of the aromatic ring), 1673 cm -1 ( C=O of the carboxylic acid groups and  C=C of the aromatic ring), 1603, 1510, and 1448 cm -1 ( C=C of the aromatic ring), 1242 cm -1 (β C-H of aromatic ring), and 1214 cm -1 (β O-H and  C-O of the aromatic ring).38-41 Ferulic acid exhibited characteristic peaks (Figure 4b, red line) at 3439 cm -1 ( O-H of the hydroxyl groups), 3077-2514 cm -1 ( O-H of the carboxylic acid groups and  C-H of the aromatic ring), 1690 cm -1 ( C=O of the carboxylic acid groups and  C=C of the aromatic ring), 1618 cm -1 ( C=C of the aromatic ring and alkenes), 1515 cm -1 ( C=C of the aromatic ring), 1276 cm -1 (β C-H and  C-O of the alcohol groups), and 1035 cm -1 ( O-CH3 ). 42-47 Sinapic acid displayed main characteristic peaks (Figure 4c, green line) at 3372 and 3245 cm -1 ( O-H of the hydroxyl groups), 3081-2846 cm -1 ( O-H of the carboxylic acid groups and  C-H of the aromatic ring), 1697 cm -1 ( C=O of the carboxylic acid groups), 1617 and 1518 cm -1 ( C=C of the aromatic ring), 1461 cm -1 ( C=C of the aromatic ring and β O-CH3 ), 1368 cm -1 ( C=C of the aromatic ring), 1247 cm -1 ( C-H ), 1209 cm -1 ( C-O and β O-H ), and 1115 cm -1 (β C-H of the aromatic ring). 39,48-50 Then, the chitosan spectrum displayed characteristic peaks (Figure 4d, black line) at 3425 cm -1 ( O-H overlapped with  N-H ), 2922 and 2870 cm -1 ( C-H ), 1641 cm -1 ( C=O of the acetamide and amine II in the primary alcohol groups), 1603 cm -1 ( N-H of the amine I group), 1420 cm -1 ( CH2 ), 1383 cm -1 ( OH of the primary hydroxyl groups), 1317 cm -1 ( C-N of the amine III in the methyl carbamyl groups), 1260 cm -1 ( OH of the hydroxyl groups), 1153 cm - as C-O-C ), 1078 cm -1 ( s C-O-C ), 1031 cm -1 ( C-O ), and 894 cm -1

Figure 4 :

 4 Figure 4: FTIR absorbance spectra of starting materials: a) coumaric acid (CA); b) ferulic acid (FA), c) sinapic acid (SA) and d) chitosan (CS). The arrows represent the characteristic maximum absorbance peaks.

Figure 3 :

 3 Figure 3: UV-VIS absorbance spectra obtained on the film forming solution after the grafting reaction (followed by dialysis) of lignin monomers onto chitosan (CS: chitosan, CA: coumaric acid, FA: ferulic acid, SA: sinapic acid). Chitosan spectrum in solution is also displayed for comparison purpose. The arrows point out characteristic maximum absorbance peaks.

Figure 5 :

 5 Figure 5: FTIR absorbance spectra obtained on grafted films with a) coumaric acid (CS-CA); b) ferulic acid (CS-FA), c) sinapic acid (CS-SA) and d) initial chitosan (CS) spectrum for comparison purpose. Spectra were only considered in the 1800-400 cm -1 range to better highlight differences. The arrows represent the characteristic maximum absorbance peaks.Vibrations in red refer to the differences noticed between initial chitosan film and chitosan grafted films. The dashed lines indicate the shift occurring from 1603 cm -1 (N-H of the amine I group) down to around 1560 cm -1 (N-H of the amine II group) after the grafting.

Figure 7 : 6 |

 76 Figure 7: FTIR absorbance spectrum of a) chitosan film grafted with lignin (CS-LG). FTIR spectra of starting materials: b) lignin (LG) and c) chitosan (CS), are also displayed for comparison. The arrows represent the characteristic maximum absorbance peak. Vibrations in red refer to the differences highlighted by FTIR between starting materials and chitosan film grafted with lignin. The dashed lines indicate the shift occurring from 1603 cm -1 (N-H of the amine I group) down to around 1560 cm -1 (N-H of the amine II group) after the grafting.

Figure 9 :

 9 Figure 9: Radical Scavenging Activity of chitosan film (CS) and grafted films with lignin (CS-LG) and lignin monomers (CA: coumaric acid, FA: ferulic acid, SA: sinapic acid) assesed DPPH• test in ethanol (n=3). Lines are eye-guides.

Figure 8 :

 8 Figure 8: Radical Scavenging Activity of lignin monomers assessed with DPPH• test in ethanol (n=3). Lines are eye-guides.

  Table

	Sample	Nitrogen (wt %)	Carbon (wt %)	Hydrogen (wt %)	Sulfur (wt %)	C/N Ratio	Grafted yield (%)
	Chitosan (90 % deacetylated) (CS)	6.65 ± 0.06	36.70 ± 0.89	6.58 ± 0.33	0	5.52 ± 0.15	-
	p-coumaric acid (CA)	0	65.79	4.87	0	0	-
	CS-CA film	5.67 ± 0.14	39.30 ± 0.65	6.17 ± 0.13	0	6.93 ± 0.27	13.8 ± 3.3
	Trans-ferulic acid (FA)	0	61.82	5.12	0	0	-
	CS-FA film	5.59 ± 0.24	39.85 ± 0.24	6.12 ± 0.23	0	7.13 ± 0.33	13.2 ± 3.0
	Sinapic acid (SA)	0	58.93	5.39	0	0	-
	CS-SA film
	Lignin (LG)	1.23 ± 0.08	59.1 ± 0.57	6.01 ± 0.14	0.62 ± 0.33	48.16 ±2.69	-
	CS-LG film	5.43 ± 0.20	43.27 ± 0.15	6.32 ± 0.28	0.12 ± 0.07	7.98 ± 0.29	9.7 ± 5.7

5.86 ± 0.38 40.49 ± 0.80 7.16 ± 0.15 0 6.94 ± 0.61 12.0 ± 3.1

Acknowledgements

This work was supported by the Regional Council of Bourgogne-Franche Comté and The "Fonds Européens de DEveloppement Régional (FEDER). The authors would like to thanks Marcel Soustelle for the elemental analysis performed at the technological Platform for Chemical analysis and Molecular synthesis (Institute of the Molecular Chemistry of Burgundy University and Sayens).

Conflicts of interest

There are no conflicts to declare.


Norah Alsadun

Georges Mouchaham

Vincent Guillerm

Justyna Czaban-Jóźwiak

Aleksander Shkurenko

Hao Jiang

Prashant M Bhatt

Prakash Parvatkar

Introducing Cantellation Strategy for the Design of Mesoporous Zeolite-like Metal-Organic Frameworks: Zr-sod-ZMOFs as a case study

Herein we report a series of novel mesoporous zirconium-based metal-organic frameworks (MOFs) with zeolitic sodalite (sod) underlying topology. Namely, Zr-sod-ZMOF-1 and 2 are constructed based on a novel cantellation design strategy. Distinctly, organic linkers are judiciously designed in order to promote the deployment of the 12-coordinated Zr hexanuclear molecular building block (MBB) as a tetrahedral secondary building unit, a prerequisite for zeolite-like nets. The resultant Zrsod-ZMOFs exhibit truncated rhombic dodecahedron mesopores with a diameter up to ≈ 43Å, while the pore volume of 1.98 cm 3 •g -1 measured for Zr-sod-ZMOF-1 is the highest reported experimental value for zeolite like MOFs based on MBBs as tetrahedral nodes.

Metal-organic frameworks (MOFs) are a unique class of crystalline materials, resulting from the assembly of organic and inorganic molecular building blocks (MBBs) into periodic structures. [START_REF] Eddaoudi | Systematic Design of Pore Size and Functionality in Isoreticular MOFs and Their Application in Methane Storage[END_REF] Since the end of the 90's, tremendous efforts have been dedicated to explore their properties and potential applications as drug carrier, 2 separation agents, [START_REF] Adil | Gas/vapour separation using ultra-microporous metal-organic frameworks: insights into the structure/separation relationship[END_REF] sensors, [START_REF] Kreno | Metal-Organic Framework Materials as Chemical Sensors[END_REF] etc. Among the almost 100 000 MOFs reported to date, [START_REF] Moghadam | Development of a Cambridge Structural Database Subset: A Collection of Metal-Organic Frameworks for Past, Present, and Future[END_REF] the touch of rational design is increasingly visible, thanks to the principles of reticular chemistry and the associated RCSR database. [START_REF] Yaghi | Reticular synthesis and the design of new materials[END_REF] Indeed, design strategies facilitated the assembly of MOFs with increasing complexity. If the assembly of common MBBs into MOFs based on the reticulation of edge-transitive nets is now widely applied, 1c, 7 researchers are committing noticeable efforts toward expanding the repertoire of reticular chemistry. [START_REF] Chen | Reticular Chemistry 3.2: Typical Minimal Edge-Transitive Derived and Related Nets for the Design and Synthesis of Metal-Organic Frameworks[END_REF] This is achievable by using complex ligands to deviate from "default" assemblies, [START_REF] Guillerm | Geometry mismatch and reticular chemistry: strategies to assemble metal-organic frameworks with nondefault topologies[END_REF] or by developing advanced strategies, such as pillaring, [START_REF] Chun | Synthesis, X-ray Crystal Structures, and Gas Sorption Properties of Pillared Square Grid Nets Based on Paddle-Wheel Motifs: Implications for Hydrogen Storage in Porous Materials[END_REF] supermolecular building layers [START_REF] Eubank | The Quest for Modular Nanocages: tbo-MOF as an Archetype for Mutual Substitution, Functionalization, and Expansion of Quadrangular Pillar Building Blocks[END_REF] and supermolecular building blocks, [START_REF] Nouar | Supermolecular building blocks (SBBs) for the design and synthesis of highly porous metal-organic frameworks[END_REF] in order promote the assembly into a precise underlying topology when multiple possibilities are plausible for the same basic building blocks. Interestingly, some of these design strategies are inspired by the well-known porous materials, zeolites. These so called zeolite-like MOFs (ZMOFs) are constructed from tetrahedral building blocks. [START_REF] Eddaoudi | Zeolite-like metal-organic frameworks (ZMOFs): design, synthesis, and properties[END_REF] Two main ZMOFs design approaches can be distinguished, depending on their basic elements of construction; i) based on single-metal ion (Cu 2+ ,Zn 2+ , Co 2+ , In 3+ , etc.) with functionalized imidazole (ZIFs) or imidazole dicarboxylate, leading to diverse structures with known sod, ana, rho etc. topologies, [START_REF] Liu | Molecular building blocks approach to the assembly of zeolite-like metalorganic frameworks (ZMOFs) with extra-large cavities[END_REF] alongside unprecedented ones (med) [START_REF] Liu | Template-directed assembly of zeolite-like metal-organic frameworks (ZMOFs): a usf-ZMOF with an unprecedented zeolite topology[END_REF] and ii) corner sharing of supertetrahedra constructed from trimeric building blocks (Fe 3+ , Cr 3+ , etc) and linear (MIL-101) [START_REF] Férey | A chromium terephthalate-based solid with unusually large pore volumes and surface area[END_REF] or triangular ligands (MIL-100), [START_REF] Férey | A hybrid solid with giant pores prepared by a combination of targeted chemistry, simulation, and powder diffraction[END_REF] systematically to afford mtn underlying topology. [START_REF] Horcajada | Extended and functionalized porous iron(iii) tri-or dicarboxylates with MIL-100/101 topologies[END_REF] The synthesis of ZMOFs with considerably expanded pores is a long-term pursuit and a persisting challenge, and several design strategies have been explored to expand and/or functionalize the pore system of the reported ZMOFs. [START_REF] Eddaoudi | Zeolite-like metal-organic frameworks (ZMOFs): design, synthesis, and properties[END_REF] The common approach to MOFs isoreticular expansion and/or decoration is by adjusting the metrics and functionalities of the organic linker, e.g. length and/or width elongation. [START_REF] Guillerm | A supermolecular building approach for the design and construction of metal-organic frameworks[END_REF] However, this strategy is less effective for obtaining highly-porous ZMOFs. 14c This is due to the fact that there are hundreds of zeolitic topologies based on linking tetrahedral building units. Besides, linker expansion/elongation can induce slight modification in the specific angles and the positioning of the coordinating functional groups, with respect to the inorganic building block, prohibiting the isoreticulation of a specific ZMOF platform. It mostly affords amorphous solids or crystalline materials based on the "default" structure for the assembly of tetrahedral building units, dia underlying topology. [START_REF] Zaworotko | Crystal engineering of diamondoid networks[END_REF] In this context, the targeted frameworks must meet specific arrangement of tetrahedral nodes at certain range of angles at the MBB level to reach the desired zeolitic net. As a semiregular edge-transitive net with simple tilling (transitivity 1121), the sodalite (SOD, sod) net has the highest occurrence, placing it as suitable target for the design of expanded ZMOFs. [START_REF] Park | Exceptional chemical and thermal stability of zeolitic imidazolate frameworks[END_REF] Markedly, the reticulation of the sod net with expanded bridging linkers will permit access to open ZMOF structures with unique characteristics, due to the fact that the sod net is not self-dual and not prone to self-interpenetration, namely a high pore/aperture size ratio, large pore volume and contracted pore-aperture size, suitable for molecular entrapment for potential applications. 13, 14b In the present work, we introduce a cantellation design approach for the conception and directed assembly of novel mesoporous ZMOFs based on zirconium MBBs.

In order to successfully and rationally design a novel generation of isoreticular ZMOFs, we identified three main requirements: i) Achieving the formation of a tetrahedral MBB; ii) Preventing the formation of dia net, the most regular edgetransitive net, considered as the default topology for the assembly of tetrahedra; [START_REF] Delgado Friedrichs | Threeperiodic nets and tilings: regular and quasiregular nets[END_REF] iii) An assembly mode that allows the tuning of the framework through ligand expansion and/or decoration. After a careful survey of existing structures and topologies, we reasoned that an efficient method to deviate from the default dia net while assembling tetrahedral nodes would be to propose a solution that prevents their stacking in staggered fashion. For this purpose, we propose a cantellation strategy (Figure 1): second order truncation of a tetrahedron, adding a face at each edge and vertex of a tetrahedron, affords the formation of cuboctahedron. Applying now the same cantellation to a couple of tetrahedra stacked in an eclipsed fashion results in triply bridged cuboctahedra stacked in an eclipsed conformation. At this stage the triple bridges between the cuboctahedra prevents any rotation and therefore "locks" them in the intended eclipsed fashion.

Cuboctahedral (cuo) MBBs are well known in reticular chemistry, with the ubiquitous Zr hexanuclear cluster of UiO-66. [START_REF] Cavka | A new zirconium inorganic building brick forming metal organic frameworks with exceptional stability[END_REF] Therefore, Zr represents a natural candidate for further design of a novel class of ZMOFs. After identifying the suitable inorganic MBB, we embarked in the design of an organic ligand that would promote the formation of the necessary triple bridge between the cuo MBBs (Figure 1b,c). If such an example has been recently reported with linear terephthalic acid in an UiO-66 polymorph with hex-topology, [START_REF] Perfecto-Irigaray | Zr 6 O 4 (OH) 4 (benzene-1,4-dicarboxylato) 6 ] n : a hexagonal polymorph of UiO-66[END_REF] this conformation can be regarded as an exception as it is widely accepted that the favored topology would be fcu, exemplified by plethora examples. [START_REF] Cavka | A new zirconium inorganic building brick forming metal organic frameworks with exceptional stability[END_REF][START_REF] Guillerm | A zirconium methacrylate oxocluster as precursor for the lowtemperature synthesis of porous zirconium(IV) dicarboxylates[END_REF] To deviate from the default fcu topology, one must modify the geometry of the ligand, i.e. introducing a geometry mismatch. [START_REF] Guillerm | Geometry mismatch and reticular chemistry: strategies to assemble metal-organic frameworks with nondefault topologies[END_REF] Indeed, systematic explorations have shown that topological diversity can be achieved through the use of bent [START_REF] Bon | Zr-and Hf-Based Metal-Organic Frameworks: Tracking Down the Polymorphism[END_REF] or zigzag 19c ligands, as well as introducing steric hindrance to alter the orientation of the carboxylates. [START_REF] Yuan | Sequential Linker Installation: Precise Placement of Functional Groups in Multivariate Metal-Organic Frameworks[END_REF] To meet our needs on the quest for the discovery of highly porous Zr-MOFs based on zeolite topologies, the ideal ligand should be: i) A ditopic linker, with a bent geometry allowing the triple bridging of MBBs without excessive constraint on the Zr-carboxylate bonds; and ii) the two carboxylates preferably oriented orthogonally to the plan of the ligand. Separately, these two requirements would not be sufficient to achieve the envisioned assembly. Indeed, controlling only the bending angle on planar ligands afforded wide topological diversity, allowing the formation of MOFs with fcu, 28 reo, 29 bon, 26 bct, 30 hbr, 31 kag 32 or pcu topologies. [START_REF] Xie | Metal-Organic Frameworks for the Capture of Trace Aromatic Volatile Organic Compounds[END_REF] In parallel, introducing steric hindrance on linear ligands favored the assembly of MOFs with bcu topology. [START_REF] Yuan | Sequential Linker Installation: Precise Placement of Functional Groups in Multivariate Metal-Organic Frameworks[END_REF] The insightful combination of these two geometrical requirements guided the design and synthesis of benzene-1,3-dimesitylenic acid (BDM, L1, Figure 1b). The ligand is based on a terphenyl core, where the central phenyl group is decorated by two benzoic groups in meta positions. To fulfill the second criterion, we introduced steric hindrance by the means of methyl groups on the ortho positions (in respect to the central phenyl group) of each of the benzoic groups. In this way, introducing the methyl groups at these specific positions will act as a lock, limiting the rotation of the rings, and keeping the peripheral carboxylate groups orthogonal to the plan of the ligand. This will overall minimize the coordination options, favors the triple bridging of the MBBs, virtually reducing their net-connectivity (points of extension) from 12 to 4: Each 12 coordinated cluster is surrounded by 4 others in a tetrahedral fashion, as a result of the 12 ligands split into four triads converging to the same neighboring cluster (Figure 2c).

Reaction of ZrCl 4 and L1 in presence of DMF and acetic acid resulted in the isolation rhombic dodecahedral crystals (Figure S16-a). Single-crystal X-ray diffraction studies revealed that, the obtained MOF encloses fully coordinated clusters (12-c) and the overall MOF has the targeted underlying sod topology, observed for the first time in M IV -based MOFs. Due to the triple bridging of the clusters by the ligands, a resulting cavity is not represented by the classic tiling of the sod topology. It is reflected by the unprecedented augmented net, csd (cantellated sodalite), unveiled thanks to our design approach. It differs from sod-a net: it also allows to appreciate the presence and connectivity mode of the cuboctahedral building blocks, where a trigonal prism completes the tridimensional tiling (Figures S21-23). Zr-sod-ZMOF-1 crystallizes in the Im-3m cubic space group with a unit cell parameter of 55.199 (7) Å and a volume of 168190(60) Å [START_REF] Adil | Gas/vapour separation using ultra-microporous metal-organic frameworks: insights into the structure/separation relationship[END_REF] . The overall framework contains two types of cages: the large truncated rhombic dodecahedral cage typical from the sodalite net is mesoporous, with a diameter ≈43Å, accessible through square windows of ≈8Å and hexagonal windows of ≈21Å apertures. In addition, the structure encompasses microporous cages with a diameter of ≈6Å, resulting from the cantellation strategy, and delimited by the three ligands bridging adjacent clusters (Figure S19-a). Each unit cell of Zr-sod-ZMOF-1 contains 3672 atoms, ca. 13.3 and 102 times more than the unit cells of ZIF-8 (276 atoms) and the sodalite zeolite (36 atoms), respectively. From a purely geometrical view, the assembly of eclipsed tetrahedra through our cantellation strategy should give an ideal T-X-T angle of 180 ° between the tetrahedral MBBs. There is however no net allowing such perfect orientation of eclipsed tetrahedra.

Therefore, mtn or mgz-x-d nets, with T-X-T angles of 175 ° and 174.5 °, respectively, could be the expected topologies. [START_REF] O'keeffe | Tetrahedral frameworks TX 2 with T-X-T angle=180°: Rationalization of the structures of MOF-500 and of MIL-100 and MIL-101[END_REF] Interestingly, in the present case, the sod net is favored, with an experimental T-X-T angle of 173.5 ° (Figure S18-c). This can be explained by its higher regularity: compared to the trinodal mtn and heptanodal mgz-x-d nets, the sod net is edge-transitive, uninodal.

A careful look at the structure permits an interesting observation: Zr-sod-ZMOF-1 contains two crystallographically independent ligands, both present in each of the triple bridge between the clusters. The experimental bending angles of 2x 112.2 ° and 1x 127 °, away from the ideal 120 ° expected for this type of bending (Figure S18-a). This observation led us to conclude that thanks to the slight The porosity of Zr-sod-ZMOF-1 has been examined by argon sorption experiment carried out at 87 K, exhibiting a type-IV isotherm, characteristic of a mesoporous material with permanent porosity (Figure 3-a). The apparent Brunauer-Emmett-Teller (BET) surface area was estimated to be 1565 m 2 •g -1 . The experimental total pore volume was calculated to be 1.98 cm 3 •g -1 , in agreement with the theoretical value (2.4 cm 3 •g -1 ). This experimental pore volume is the highest for sod-ZMOFs, and even for ZMOFs based on 4-c tetrahedral MBBs (Figure 3-c). [START_REF] Yang | Principles of Designing Extra-Large Pore Openings and Cages in Zeolitic Imidazolate Frameworks[END_REF] The pore size distribution shows a sharp pore size at 43Å, in agreement with the crystallographic data for mesopores. Since the primary pore system in Zr-sod-ZMOF-1 is mesoporous, the model deployed could not assess the contribution from the micropores. The micropores between the clusters generated by the cantellation with 3 bent ligands can be relatively estimated by using only lowpressure data points (up to P/P 0 = 0.03), asserting the presence of micropores around 6.2Å and their contribution to the overall porosity (Figure S24-c). In order to assess the strength of our cantellation design approach, we intended to prepare isoreticular Zr-sod-ZMOFs (Figure 4) and a novel ligand was designed. 4,4'-(2,4,6trimethylpyridine)dibenzoic acid (TMPDB, L2), has the same angular characteristics as L1, however, its steric hindrance is now located in the central part of the ligand, on a pyridine ring. As expected, the use of ligand L2 allowed to obtain an isoreticular analogue to Zr-sod-ZMOF-1 (Tables S1-2). The phase purity was further confirmed by powder X-ray diffraction pattern (PXRD, Figure S17). Due to the similarities between L1 and L2, Zr-sod-ZMOF-2 exhibit same type of cages. The large truncated rhombic dodecahedron mesopore cage, having a diameter of about 43.5Å and a confined cage between the triple ligands of 4-6Å, with two windows: square with 12Å and hexagon with 22Å (Figure S19-b). Similar to Zr-sod-ZMOF-1, there are two crystallographically independent ligands in the Zr-sod-ZMOF-2: 2x 118 °, 1x 130.4 ° and the tetrahedral units show a T-X-T angle of 173 ° (Figure S18-a), In this structure, the dihedral angles between the benzoic moieties and the central core of the ligand are maintained at ca. 90 °, "locked" by the steric hindrance. In summary, we have developed a cantellation design approach that afforded the deployment of the 12-coordinated Zr-based MBB as a tetrahedral secondary building unit. Locked in eclipsed conformation through triple bridging by the organic ligands, the stacking of the MBBs prevents the formation of interpenetrated MOFs with dia topology while allowing the formation of zeoliticnets. Combined with appropriate ligand design, this strategy permitted the discovery of two isoreticular mesoporous Zr-sod-ZMOFs, the first examples of Zr-MOFs with zeolitic topology. Zrsod-ZMOF-1 has the highest experimental pore volume reported for any sodalite type material so far (≈3.5 times higher than ZIF-8), as well as for any zeolitic material based on tetrahedral MBBs. Finally, our cantellation strategy opens new opportunities for the deliberate use of highly coordinated clusters as secondary building units with lower connectivity, through multiple bridging.

Figure 1 .

 1 Figure 1. a) Schematic of the tetrahedra cantellation strategy leading to multiple bridges, allowing the building units to be "locked" in eclipsed conformation and b) design of a sterically hindered bent ligand, locking the carboxylic group in the appropriate position to c) form a triple bridge between the MBBs.

Figure 2 .

 2 Figure 2. Illustration of the bottom up construction of Zr-sod-ZMOF-1 (center); a) organic and inorganic MBBs assemble through b) the formation of triple bridges. c) Each inorganic MBB is connected to four others in a tetrahedral fashion. d) The tetrahedral MBBs are oriented in an eclipsed fashion, with e) a slight tilt characteristic of zeolites, allowing the overall formation of f) the sodalite type cage.

Figure 3 .

 3 Figure 3. a) Type 4 Ar adsorption-desorption isotherm , b) the corresponding PSD and c) Experimental pore volume of Zr-sod-ZMOF-1 as compared to the highest reported values from frameworks based on tetrahedral nodes and with sod topology (ZIF-8 and In-sod-ZMOF) in gold. Experimental pore size distribution of Zr-sod-ZMOF-1 showing the highest reported experimental value among the same set of frameworks in blue. d) Cage size difference in Zeolitic SOD structure, ZIF-8 and Zr-sod-ZMOF-1.

Figure 4 .

 4 Figure 4. The relocation of the steric hindrance from a) the arms of the ligands (Zr-sod-ZMOF-1) to b) the central core allows the formation of an isoreticular analogue, Zr-sod-ZMOF-2.

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ACKNOWLEDGMENT

Research reported in this publication was supported by the King Abdullah University of Science and Technology (KAUST). G.M and M.E thank Aramco sponsored research fund (contract. 66600024505) ACS Paragon Plus Environment Journal of the American Chemical Society

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Supporting Information

The Supporting Information is available free of charge on the ACS Publications website at DOI:. Synthesis, PXRD, TGA, and additional sorption data (PDF)

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Corresponding Author *E-mail for M.E.: mohamed.eddaoudi@kaust.edu.sa. Author Contributions ‡These authors contributed equally.

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G. M. is now affiliated to

Notes

The authors declare no competing financial interests.

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