Tracey L Hammer

Pierre Bize

Benoit Gineste

Jean-Patrice Robin

René Groscolas

Vincent A Viblanc

Disentangling the "many-eyes", "dilution effect", "selfish herd", and "distracted prey" hypotheses in shaping alert and flight initiation distance in a colonial seabird

Keywords: antipredator behaviour, escape flight distance, optimal escape decisions, predation risk, predator-prey decision, risk taking

Group living is thought to have important antipredator benefits for animals, owing to the mechanisms of shared vigilance ("many-eyes" hypothesis), risk dilution ("dilution effect" hypothesis), and relative safety in the center of the group ("selfish herd" hypothesis). However, it can also incur costs since social stimuli, such as conspecific aggression, may distract individuals from anti-predator behavior ("distracted prey" hypothesis). We simultaneously evaluated how these four different hypotheses shape anti-predator behaviors of breeding king penguins (Aptenodytes patagonicus), which aggregate into large colonies, experience frequent aggressive social interactions, and are regularly exposed to predation by giant petrels (Macronectes sp.) and brown skuas (Catharacta loonbergi) when breeding on land. We approached 200 incubating penguins at four different periods of the breeding season across a range of overall increasing colony densities. We measured the distance at which focal birds detected the approaching threat (alert distance: AD), whether birds decided to flee or not, and the distance of flight initiation (flight initiation distance: FID, viz. the bird attempting to walk away with its egg on its feet). We quantified relative local neighbor density, centrality within the colony (rank), and the number of aggressions the focal bird emitted towards neighbors during the approach. We found that birds engaged in aggressive conflicts with neighbors were less likely to flee, and that increasing relative local neighbor density at low and medium overall colony density resulted in a decrease in bird AD, both supporting the "distracted prey" hypothesis. However, at maximal overall colony density, increasing relative local neighbor density resulted in longer AD, supporting the "many-eyes" hypothesis. We found no support for the "dilution effect" and "selfish herd" hypotheses, and no effects of any hypothesis on FID.

INTRODUCTION

Predation can be an important source of mortality in wild animals [START_REF] Roos | A review of predation as a limiting factor for bird populations in mesopredator-rich landscapes: a case study of the UK[END_REF].

Aggregating into large social groups is often viewed as a solution to decrease predation risk for the individual and its offspring [START_REF] Alexander | The evolution of social behaviour[END_REF][START_REF] Hoogland | The evolution of coloniality in white-tailed and black-tailed prairie dogs (Sciuridae: Cynomys Leucurus and C. Ludovicianus)[END_REF]. At least four non-mutually exclusive hypotheses have been proposed to explain how group size and social interactions may influence predator-prey interactions (for a review of the relationship between group size and flight initiation distance, see [START_REF] Ydenberg | The economics of fleeing from predators[END_REF][START_REF] Stankowich | Fear in animals: a meta-analysis and review of risk assessment[END_REF].

Firstly, one important mechanism identified in decreasing predation risk with increasing group size is that of shared vigilance by group members towards potential threats [START_REF] Caraco | Avian flocking in the presence of a predator[END_REF][START_REF] Lima | Behavioral decisions made under the risk of predation: a review and prospectus[END_REF][START_REF] Quenette | Functions of vigilance behaviour in mammals: a review[END_REF]. Since larger groups have more individuals scanning for predators at any given time, the presence of "many eyes" in a group allows for the earlier detection of predators than would be possible for single individuals (the "many-eyes hypothesis"; [START_REF] Pulliam | On the advantages of flocking[END_REF][START_REF] Lima | Behavioral decisions made under the risk of predation: a review and prospectus[END_REF]. Information on the presence of a predator can disseminate rapidly through the group, most often through the use of alarm calls, allowing individuals to assess the risk and decide to flee sooner than is possible alone [START_REF] Burger | Human distance and birds: tolerance and response distances of resident and migrant species in India[END_REF][START_REF] Mayer | Water body type and group size affect the flight initiation distance of European waterbirds[END_REF]). Thus, larger groups are often more rapid in detecting a predator than single individuals, and more likely to raise alarm of an approaching predator sooner than smaller groups [START_REF] Hoogland | The evolution of coloniality in white-tailed and black-tailed prairie dogs (Sciuridae: Cynomys Leucurus and C. Ludovicianus)[END_REF][START_REF] Van Heezik | Effect of human disturbance on beach groups of jackass penguins[END_REF][START_REF] Cresswell | Flocking is an effective anti-predation strategy in redshanks, Tringa totanus[END_REF], though vigilance behavior may also be affected by other environmental characteristics such as species gregariousness, diet or habitat [START_REF] Morelli | Contagious fear: Escape behavior increases with flock size in European gregarious birds[END_REF].

Secondly, large groups also provide the advantage of diluting individual predation risk [START_REF] Ydenberg | The economics of fleeing from predators[END_REF]. Most predatory species can only capture a single prey at a time, and thus the chance that each individual prey will be the target of predation reduces with increasing group size [START_REF] Dehn | Vigilance for predators: detection and dilution effects[END_REF][START_REF] Roberts | Why individual vigilance declines as group size increases[END_REF][START_REF] Bednekoff | Re-examining safety in numbers: Interactions between risk dilution and collective detection depend upon predator targeting behaviour[END_REF]. According to the "dilution effect hypothesis" individuals in larger groups may tolerate the closer approach of predators before initiating flight (enabling a longer amount of time to be spent on tasks such as foraging), since the risk of predation is diluted with each additional conspecific nearby [START_REF] Cresswell | Flocking is an effective anti-predation strategy in redshanks, Tringa totanus[END_REF][START_REF] Boland | An experimental test of predator detection rates using groups of free-living emus[END_REF]; [START_REF] Fernández-Juricic | Do variations in scanning behavior affect tolerance to human disturbance?[END_REF]. The dilution effect is not only limited to larger group sizes, but also to denser groupings of individual prey, regardless of overall group size [START_REF] Roberts | Why individual vigilance declines as group size increases[END_REF][START_REF] Hebblewhite | Effects of elk group size on predation by wolves[END_REF][START_REF] Frommen | Disentangling the effects of group size and density on shoaling decisions of three-spined sticklebacks (Gasterosteus aculeatus)[END_REF]. For example, many bird species that migrate in flocks or cluster together on breeding grounds may benefit from the dilution effect by reducing individual predation risk while foraging or breeding [START_REF] Wiklund | Natural selection of colony size in a passerine bird[END_REF][START_REF] Harts | Predation can select for later and more synchronous arrival times in migrating species[END_REF][START_REF] Duca | Predator search image and the dilution effect: when is the best time to nest[END_REF], though in some groups individual risktaking may vary with life-history stage (e.g., in long-distance migratory waders, Charadriiformes, risk-taking is lowest during reproduction and highest during migration; [START_REF] Mikula | Adjusting risk-taking to the annual cycle of longdistance migratory birds[END_REF].

Thirdly, individual spatial positioning within the group may provide dissimilar antipredator benefits. The "selfish herd" hypothesis [START_REF] Hamilton | Geometry for the selfish herd[END_REF] suggests that animals located more centrally in a group should be less vulnerable to attacks from predators than those on the periphery as predators come from outside the group and target the closest available prey [START_REF] Hamilton | Geometry for the selfish herd[END_REF]. In other words, maintaining conspecifics between a predator and oneself will reduce individual predation risk. Prey animals should therefore select higher value central positions when joining a group, which is indeed seen in minnows, Phoxinus phoxinus [START_REF] Krause | the effect of "Schreckstoff" on the shoaling behaviour of the minnow: a test of Hamilton's selfish herd theory[END_REF], sheep, Ovis aries [START_REF] King | Selfish-herd behaviour of sheep under threat[END_REF], and fiddler crabs, Uca pugilator [START_REF] Viscido | Quantitative analysis of fiddler crab flock movement: evidence for "selfish herd" behaviour[END_REF]. If selfish herd effects are important, there should be evidence of divergent predation rate, pressure or risk between the center and the periphery of a group [START_REF] Morton | The selfish herd revisited: do simple movement rules reduce relative predation risk?[END_REF][START_REF] Viscido | The response of a selfish herd to an attack from outside the group perimeter[END_REF]. When redshanks, Tringa totanus, were targeted by sparrowhawks, Accipiter nisus, birds that were more peripheral and further from neighbors were at the highest risk of predation [START_REF] Quinn | Testing domains of danger in the selfish herd: sparrowhawks target widely spaced redshanks in flocks[END_REF]. In colonial web-building spiders, Meteperira incrassata, predatory attacks occur most often on the periphery [START_REF] Rayor | Trade-offs in foraging success and predation risk with spatial position in colonial spiders[END_REF]. There may also be evidence of different levels of investment by prey into anti-predator monitoring between central and peripheral animals. Increased vigilance by peripheral animals has been observed in black-tail prairie dogs, Cynomys ludovicianus [START_REF] Hoogland | The evolution of coloniality in white-tailed and black-tailed prairie dogs (Sciuridae: Cynomys Leucurus and C. Ludovicianus)[END_REF], African mammals [START_REF] Burger | Vigilance in African mammals: differences among mothers, other females, and males[END_REF], including springboks, Antidorcas marsupialis, [START_REF] Bednekoff | Vigilance in Nxai Pan springbok, Antidorcas marsupialis[END_REF], and scaled doves, Columbina squammata [START_REF] Dias | Effects of position and flock size on vigilance and foraging behaviour of the scaled dove Columbina squammata[END_REF].

Finally, although larger groups provide the benefit of shared vigilance and predation risk dilution, individuals in such groups have to process a noteworthy amount of social information from their conspecifics [START_REF] Treves | Theory and method in studies of vigilance and aggregation[END_REF]. As a consequence, the time spent processing social cues and interacting with conspecifics, noise and conflicts from and between conspecifics may detract from the time and energy investments usually placed into scanning for predators [START_REF] Mooring | Costs of allogrooming in impala: distraction from vigilance[END_REF][START_REF] Yee | The costs of conspecifics: are social distractions or environmental distractions more salient?[END_REF]. This is known as the "distracted prey hypothesis" which proposes that external stimulisuch as soundsare capable of hijacking finite resources, thus impairing the prey's ability to detect and react to approaching predators [START_REF] Chan | Anthropogenic noise affects risk assessment and attention: The distracted prey hypothesis[END_REF][START_REF] Petrelli | First to flush: the effects of ambient noise on songbird flight initiation distances and implications for human experiences with nature[END_REF]. Social distractions (such as aggressive interactions between conspecifics) may also distract prey from predator detection and delay behavioral responses such as the initiation of flight. This is, for instance, the case in a cichlid fish species (Neolamprologus pulcher), where individuals respond significantly later to predator images during territorial contests than when they are alone, illustrating the trade-off between time and energy investments into aggressive social behaviors and vigilance towards predators [START_REF] Hess | Territorial aggression reduces vigilance but increases aggression towards predators in a cooperatively breeding fish[END_REF].

Here, we investigated the relative importance of the (1) "many-eyes", ( 2) "dilution effect", (3) "selfish herd" and (4) "distracted prey" hypotheses on prey anti-predator behaviors of breeding king penguins (Aptenodytes patagonicus). These four hypotheses are not mutuallyexclusive, and thus can be investigated simultaneously. For instance, individuals in larger groups may benefit from risk-dilution, shared vigilance, and selfish herd effects, all while paying potential costs of social distractions. King penguins are an ideal model system to test these nonmutually exclusive alternatives. This seabird species forms extensive, densely packed colonies when breeding on land (over tens of thousands of birds strong, [START_REF] Barbraud | Population trends of penguins in the French Southern Territories[END_REF]. Breeding birds incubate/raise a single egg/chick on their feet and aggressively defend their small breeding territory against both predators and conspecifics [START_REF] Stonehouse | The king penguin Aptenodytes patagonica of South Georgia. Falklands Islands Dependencies Survey[END_REF][START_REF] Weimerskirch | The breeding biology and population dynamics of King Penguins Aptenodytes patagonica on the Crozet Islands[END_REF].

Aggressive social interactions are particularly high and can average 100 interactions per hour [START_REF] Côté | Aggressiveness in king penguins in relation to reproductive status and territory location[END_REF]. Of particular interest is the finding that king penguins are highly sensitive to social aggression, even when they are not directly involved in social conflicts (as demonstrated by elevated by-stander heart rate responses when witnessing aggressive neighbors fighting; Viblanc et al. 2012). In addition, higher stress hormones and heart rates are detected in more densely packed colony areas (Viblanc et al. 2014a;Viblanc et al. 2014b). Thus, frequent social aggression in these birds may interfere with antipredator behaviors, such as vigilance and flight.

King penguins are subject to on-land predation by giant petrels, Macronectes giganteus and Macronectes halli, and brown skuas, Catharacta loonbergi, especially known to predate on eggs and young chicks, but occasionally targeting adults as well [START_REF] Hunter | The impact of avian predator scavengers on king penguin Aptenodytes patagonicus chicks at Marion Island[END_REF][START_REF] Descamps | Impact of predation on king penguin Aptenodytes patagonicus in Crozet Archipelago[END_REF]. These predators will harass incubating and brooding adults sometimes causing them to flee and abandon their eggs. Breeding birds then face three choices: to stay and fight, risking injuries that could be fatal; to flee cumbersomely by waking with their egg or offspring on the top of their feet and to cluster closer to neighbors; or to abandon reproduction and flee entirely by walking rapidly (often includes tripping and flipper beating against the ground; RG, VAV, JPR, BG, PB, TH; personal observations). Yet, how shared vigilance (1. "many-eyes" hypothesis), safety in large numbers (2. "dilution effect" hypothesis), safety in a central location (3. "selfish herd"), and acute social interactions (4. "distracted prey" hypothesis) (Table 1) interact in shaping prey antipredator behavioral responses is unknown.

We used Alert Distance (AD), or the distance at which the targeted bird detects an approaching threat (in this case, an experimenter), and Flight Initiation Distance (FID), or the distance at which the targeted bird begins to flee from the approaching threat to simultaneously investigate the "many-eyes", "dilution effect", "selfish herd" and "distracted prey" hypotheses.

AD and FID are popular measures used in the literature to study the reactions of prey to approaching danger and optimal escape strategies. The great advantage of these measures is that they can be collected through non-lethal approaches by a human experimenter using standardized approach protocols (see [START_REF] Hammer | Repeatability of alert and flight initiation distances in king penguins: effects of colony, approach speed, and weather[END_REF] for an example in king penguins). From an evolutionary perspective, prey are expected to react in much the same way to a human approach as to an approach by a true predator, as behavioral responses should be selected to over-estimate rather than under-estimate risk [START_REF] Frid | Human-caused disturbance stimuli as a form of predation risk[END_REF][START_REF] Beale | Human disturbance: People as predation-free predators?[END_REF]. AD and FID are therefore promising behavioral responses that can be used to better understand how group size and social interactions may shape predator-prey interactions.

We evaluated AD and FID in response to standardized human approaches in relation to both overall colony density (low, med, high, maximal) that increased as the breeding season progressed, relative local neighbor density (number of neighbors around a focal bird centered around the mean overall colony density at any given stage of the season), relative colony rank (rank centered with each colony density stage), and the number of aggressive interactions (i.e., social distractions) exchanged between the focal bird and its neighboring conspecifics during the approach. Whereas the "many eyes" hypothesis predicts that AD should occur earlier in larger groupings as there are more vigilant individuals present at any given point in time, the "distracted prey" hypothesis predicts that AD and/or FID will be delayed in denser groups due to increased social stimuli, such as aggression with neighbors, that hinders predator detection and flight. The "selfish herd" hypothesis predicts that peripheral individuals in the colony (those of low colony rank as counted from the periphery to the center of the colony) where predation risk is highest, will have higher AD and FID than individuals in less risky central positions. Finally, the "dilution effect" predicts that FID will be delayed at high local social density due to reduced predation risk on the individual, but should be unrelated to social interactions (social aggression, in our case). Predators may be allowed to approach even closer in aggressive groupings of prey than the dilution effect predicts alone, as is the case in king penguins, as larger groups may allow for group defense from predators (i.e., mobbing).

METHODS

Study site and animals

During the 2011-2012 breeding season, 200 king penguins were randomly chosen and approached in "La Baie du Marin" colony (Possession Island, Crozet Archipelago, 46°25'S -51°52'E), home to approximately 22 000 breeding pairs [START_REF] Barbraud | Population trends of penguins in the French Southern Territories[END_REF]. According to STRANGE guidelines [START_REF] Webster | How STRANGE are your study animals?[END_REF], study subjects were selected to reduce potential biases (and were hence not blindly selected), nevertheless we highlight some of the potential biases here. All birds were incubating and therefore of similar breeding status (but see discussion) and appeared in physically good overall condition; but we did not have any information on bird age, sex, laying date, or previous exposure to humans. Individuals were chosen from a distance and not after being approached or captured, therefore we expect no strong initial sampling bias. Between successive approaches, birds were at least 50 meters apart. The

Approach protocol

Approaches (always the same experimenter, BG, dressed in the same clothing) were started at a standardized start distance of 18 m when the focal individual was awake. A distance of 18 m corresponded to a distance 1.5 times greater than maximal detection distance recorded in a preliminary study to ensure starting distance far exceeded maximum alert distance [START_REF] Hammer | Repeatability of alert and flight initiation distances in king penguins: effects of colony, approach speed, and weather[END_REF], see also [START_REF] Fleming | Scavenging opportunities modulate escape responses over a small geographic scale[END_REF]. Start distance was standardized due to its known effect on AD and FID [START_REF] Blumstein | Flight-initiation distance in birds is dependent on intruder starting distance[END_REF][START_REF] Blumstein | Flush early and avoid the rush: a general rule of antipredator behavior?[END_REF][START_REF] Dumont | Flight initiation distance and starting distance: biological effect or mathematical artefact?[END_REF]. Distances were measured to the nearest cm using a laser telemeter (Leica DISTO TM D5 Lasermeter, Leica Geosystems AG, Hexagon, Sweden). The experimenter, while walking at a regular speed, followed a direct trajectory towards the focal individual. Both when the first sign of detection was observed (AD; focal animal tilting its head or stretching its neck in the direction of the experimenter), and when flight was initiated (FID; focal animal attempting to walk away with its egg on its feet), the experimenter took a standardized one-minute pause to record the distance between his position and the bird. Several focal birds did not flee up until contact. Thus, FID was divided into two separate analyses: i) the decision to flee or not, and ii) the flight initiation distance for birds that decided to flee. During the approach, the number of aggressive interactions initiated by the focal bird towards its neighbors was counted by the approaching experimenter and recorded as a proxy of social distraction. Aggressive interactions included: bill gaping (non-physical-threats displays, the focal bird extending its head with bill open wide, vocalizing towards a neighbor); bill pecking (the birds extending its neck and physically pecking a neighbour) and flipper blows (the bird striking a neighbor with an extended flipper) (see [START_REF] Côté | Aggressiveness in king penguins in relation to reproductive status and territory location[END_REF], Viera et al. 2011). The number of aggressive interactions initiated by the focal bird was then weighted to account for known differences in energetics: aggressions with physical contact (bill pecking, flipper blows) weighed more heavily than non-physical threats (bill gaping); according to Viera et al. (2011), contact aggressions are 3.2 times more energy costly than threats (mean  SE = 0.69  1.00, range = 0.0 -5.2).

Two speeds of approach (prior to AD and prior to FID) were calculated using the distance walked (m) and the duration of approach (s). The mean  SE speed of approach prior to AD was 0.51  0.07 m/s (range = 0.28 -0.76 m/s), and between AD and FID was 0.42  0.05 m/s (range = 0.24 -0.54 m/s). Weather conditions were recorded prior to each approach and used as control variables in the analyses [START_REF] Hammer | Repeatability of alert and flight initiation distances in king penguins: effects of colony, approach speed, and weather[END_REF]. Air temperature was recorded to the nearest 1°C, and wind and rain levels were scored between 0 (none) and 2 (strong or heavy), intermediate levels (0.5) being allowed.

Before approaching the birds, we counted the number of neighbors surrounding each focal individual, defined as the first circle of animals that were direct neighbors (mean  SE = 7.49  1.93; range = 4 -14) and recorded the mean distance of the neighbors to the focal individual (mean  SE = 0.81  0.14; range = 0.53 -1.22 m). The distance between the birds was initially visually estimated from a distance and subsequently corrected to the closest 10 cm during the approach. Local density (number of birds/m 2 ) was then calculated as the number of neighbors surrounding a focal bird divided by the area of a circle (πr 2 ) with the mean distance between the focal bird and its neighbors acting as the radius. Local bird density increased with overall colony density (F 3,192 =28.0, P<0.001) (Fig. 1). As local neighbor density naturally increased together with increasing colony density (i.e., low, medium, high, maximal) as the breeding season advanced, we centered local neighbor density within each colony density stage to obtain an index of local neighbor density which was independent of overall colony density. Thus, relative local neighbor density was calculated as d -, with d being the local neighbor density of a focal bird, and being the mean neighbor density over all birds measured at a given stage (i.e., within low, medium, high, and maximal colony density stages). A bird with relative local neighbor density >0 (or <0) thus experienced a "local" social environment that was relatively more or less crowded than the average at a given stage.

Statistical analyses

All statistical analyses were performed in R 1.4.1106 (R Development Core Team 2021). Results are presented as means  SE. Effects were considered statistically significant for P < 0.05. We investigated the "many eyes", "dilution effect", "selfish herd", and "distracted prey" hypotheses by considering the joint effects of overall colony density, relative local neighbor density, relative colony rank (see below), and number of aggressive interactions of the focal individual to their neighbor (all independent variables) on focal bird AD, FID, and decision to flee or not (all dependent variables in separate models). The number of aggressions displayed against neighboring individuals during the entire approach was used to quantify the amount of social distraction to which the focal birds were subject. Similar to relative local neighbor density, we calculated relative colony rank (or the relative centrality of the focal bird within the colony) by centering bird rank within each overall colony density stage (low, medium, high, maximal) in the manner of r -, with r being the rank of a focal bird, and being the mean rank over all measured birds at a given stage. Thus, a bird with a relative colony rank >0 (or <0) was relatively more (or less) central in the colony than the average at a given stage. General linear mixed models with appropriate error distributions were used to investigate sources of variation in AD (gaussian distribution), the decision to flee or not (binomial distribution), and FID (gaussian distribution after log transformation). Models were run using the package "lme4" functions lmer and glmer [START_REF] Bates | Fitting linear mixed-effects models using lme4[END_REF]. Models were checked for the normality of their residuals using "fitdistrplus" (Delignette-Muller and Dutang 2015). For models with a gaussian distribution, the significance of fixed effects were tested with "lmerTest" anova [START_REF] Kuznetsova | lmerTest Package: Tests in Linear Mixed Effects Models[END_REF]) using F tests with Satterthwaite estimation for the denominator degree of freedom. Wald chi-square tests were used for models with a binomial distribution (Anova). We further tested if behavioral decisions (AD, FID, and the decision to flee or not) were affected by the interaction between relative local neighbor density and overall colony density (low, medium, high, maximal); this interaction was dropped from the models if it was non-significant. The area of the colony in which we approached the bird was initially included as a random factor in all our models to control for unmeasured differences in landscape, chronic and spatially variable amounts of human disturbance, etc., and the fact that several birds were measured in similar areas. However, this random effect was removed from the binomial model as no variation in the decision to flee could be attributed, and the model could not converge. In all models we initially controlled for time of day (linear and quadratic effects, decimal), weather (temperature, wind and rain levels), and speed of approach to account for tentative effects on AD, FID, and the decision to flee [START_REF] Hammer | Repeatability of alert and flight initiation distances in king penguins: effects of colony, approach speed, and weather[END_REF]), but removed them from the final models if they were non-significant (all P > 0.097). All independent variables were tested for collinearity using Variance Inflation Factors (VIF) according to the suggestions by [START_REF] Zuur | A protocol for data exploration to avoid common statistical problems[END_REF] and all variables were scaled and centered prior to inclusion [START_REF] Schielzeth | Simple means to improve the interpretability of regression coefficients[END_REF]. Temperature was, in any case, removed from all the models due to collinearity issues with colony density as temperature generally increased as the summer season progressed and the breeding season advanced.

Ethics statement

No animal was caught or handled over the course of this study. The research was approved by the Ethical Committee of the Institut Polaire Français -Paul-Emile Victor. Authorization to enter the colony and approach birds was obtained from Terres Australes et Antarctiques Françaises.

The observations complied with the current laws of France. No eggs were abandoned during the course of this study. Of the 200 individuals approached, 50% did not flee. Focal birds that were more aggressive towards their neighbors during approach were significantly less likely to flee (X 2 = 4.16, P = 0.041). The decision to flee (GLM, N = 193 with complete information), was not explained by relative colony rank, relative local neighbor density, or overall colony density (all X 2 < 1.6) (Fig. 4).

RESULTS

Over

Of the individuals that decided to flee (LMM, N = 93), the mean FID was at 3.00  1.57 m; (range = 0.54 -8.46 m). Birds showed greater FID when approached at faster speeds between AD and FID (F 1,79.8 = 20.1, P <0.001) (Fig. 5). Variation in FID was not explained by aggression with neighbors (F 1,78.5 = 2.45, P = 0.122), relative colony rank (F 1,72.0 < 1), relative local neighbor density (F 1,80.0 < 1), or overall colony density (F 3,78.7 = 1.02, P = 0.390).

DISCUSSION

This study examined the contribution of the "many-eyes", "dilution effect", "selfish herd", and "distracted prey" hypotheses in shaping prey anti-predator behavior in colonial king penguins. Our results show that at low and medium overall colony density, increasing relative local neighbor density resulted in a decrease in bird alert distance (AD) to an approaching experimenter, while at maximum overall colony density, increasing relative local neighbor density resulted in longer AD. These results provide support for the "distracted prey" hypothesis at low colony densities and the "many-eyes" hypothesis at maximum colony density. In addition, birds who engaged in aggressive behaviors with their neighbors during the approach were less likely to initiate flight, supporting the "distracted prey" hypothesis. In contrast, bird flight initiation distance (FID) did not appear to vary depending on overall or relative local bird density, nor was it affected by bird aggression or relative colony rank. Our results provide limited evidence in support of a selfish herd effect or risk dilution effect in flightless colonial penguins typically exposed to both land-based (giant petrels stalking the outskirts of the colony) and airborne (brown skuas) predators.

To the best of our knowledge, no study has so far investigated all four of these hypotheses simultaneously, especially not in incubating birds, although the "many-eyes" and "dilution effect" hypotheses have been simultaneously studied using both mathematical models [START_REF] Fairbanks | Mechanisms of the group-size effect on vigilance in Columbian ground squirrels: dilution versus detection[END_REF][START_REF] Rieucau | Many eyes or many ewes: vigilance tactics in female bighorn sheep Ovis canadensis vary according to reproductive status[END_REF][START_REF] Beauchamp | Disentangling the various mechanisms that account for the decline in vigilance with group size[END_REF]) and field studies [START_REF] Boland | An experimental test of predator detection rates using groups of free-living emus[END_REF][START_REF] Schmitt | Determining the relative importance of dilution and detection for zebra foraging in mixed-species herds[END_REF]. For instance, in bighorn sheep (Ovis canadensis) foraging ewes were found in predictive models to benefit from both the "many-eyes" and "dilution effect" hypotheses as group size increased [START_REF] Rieucau | Many eyes or many ewes: vigilance tactics in female bighorn sheep Ovis canadensis vary according to reproductive status[END_REF]. Similarly, emus (Dromaius novaehollandiae) spent less time in vigilance in larger groups, detected approaching threats sooner, and waited longer before initiating flight, supporting both the many-eyes and dilution effect hypotheses [START_REF] Boland | An experimental test of predator detection rates using groups of free-living emus[END_REF]. In Columbian ground squirrels (Urocitellus columbianus) a predator detection model was better supported than a dilution model [START_REF] Fairbanks | Mechanisms of the group-size effect on vigilance in Columbian ground squirrels: dilution versus detection[END_REF], and in plains zebras (Equus quagga), predator detection benefits were found to be more influential in mixed-species groupings than dilution effects [START_REF] Schmitt | Determining the relative importance of dilution and detection for zebra foraging in mixed-species herds[END_REF]. Over 53 published studies, [START_REF] Beauchamp | Disentangling the various mechanisms that account for the decline in vigilance with group size[END_REF] found the many-eyes and dilution effect hypothesis acted together to reduce predation risk most often in species that form groupings. These studies serve to emphasize the point that hypotheses in relation to group size on prey reactions to approaching predators and other environmental (social) stimuli are not mutually exclusive and may frequently work in concert [START_REF] Roberts | Why individual vigilance declines as group size increases[END_REF]). Yet, whereas the "many-eyes" and "dilution effect" hypotheses have been considered together in past studies, the inclusion of the "distracted prey" or "selfish herd" hypotheses in understanding prey reactions remains an open question. Although these hypotheses have not been considered conjointly, all but the "distracted prey" hypothesis have been tested individual in starlings, Sturnus vulgaris [START_REF] Zoratto | Theories commonly adopted to explain the antipredatory benefits of the group life: The case of starling (Sturnus vulgaris)[END_REF]. They have been shown

in larger groups to decrease individual vigilance yet respond faster to the approach of a model predator ("many-eyes", Powell 1974), form larger flocks when predation risk is high ("dilution effect", [START_REF] Carere | Aerial flocking patterns of wintering starlings, Sturnus vulgaris, under different predation risk[END_REF], and individuals show higher rates of vigilance and decreased foraging rate when on the periphery of the group ("selfish herd", [START_REF] Jennings | Influence of position in the flock and flock size on vigilance in the starling, Sturnus vulgaris[END_REF].

When considered alone, the "selfish herd" hypothesis has mixed support in the field (for mathematical support see: [START_REF] Reluga | Simulated evolution of selfish herd behavior[END_REF][START_REF] Morrell | Spatial positioning in the selfish herd[END_REF], with some studies supporting individual risk minimization by selfish attraction to other members when under attack (sheep, [START_REF] King | Selfish-herd behaviour of sheep under threat[END_REF]fish, Krause 1993;crabs, Viscido and Wethey 2002), and other failing to detect such effects (fish, [START_REF] Parrish | Re-examining the selfish herd: are central fish safer?[END_REF]birds, Sankey et al. 2021).

Of the four hypotheses ("many-eyes", "dilution effect", "selfish herd", and "distracted prey"), only the "many-eyes", "selfish herd", and "distracted prey" hypotheses imply changes in vigilance effort. In the "many-eyes" hypothesis, larger and/or denser groupings contain more individuals that can contribute to group-vigilance at any point in time, leading to an increase in AD. Indeed, AD is expected to be longer when the focal individual is investing more into vigilance behavior, surveying its surroundings, thus being able to detect approaching threats sooner [START_REF] Fernández-Juricic | Do variations in scanning behavior affect tolerance to human disturbance?[END_REF][START_REF] Beauchamp | Vigilance, alarm calling, pursuit deterrence, and predator inspection[END_REF]Uchida et al. 2019;[START_REF] Morelli | Contagious fear: Escape behavior increases with flock size in European gregarious birds[END_REF]; but see [START_REF] Tätte | Antipredator function of vigilance re-examined: vigilant birds delay escape[END_REF]. According to the "selfish herd" hypothesis, individuals in the periphery who are less protected by conspecifics face the highest predation risk and should therefore have the highest vigilance (hence highest AD). In contrast, the "distracted prey" hypothesis predicts that external stimuli may distract from vigilance behavior, leading to shorter AD. At low and medium colony densities, we observed that incubating adult king penguins show a decrease in AD as relative local neighbor density increased supporting the "distracted prey" hypothesis. However, at maximal colony density AD increased with relative local neighbor density, consistent with the "many-eyes" hypothesis. Earlier in the breeding season (at low colony density) king penguin breeding pairs establish their territories within the colony [START_REF] Stonehouse | The king penguin Aptenodytes patagonica of South Georgia. Falklands Islands Dependencies Survey[END_REF][START_REF] Weimerskirch | The breeding biology and population dynamics of King Penguins Aptenodytes patagonica on the Crozet Islands[END_REF]. The social aggression that occurs during territory establishment [START_REF] Côté | Aggressiveness in king penguins in relation to reproductive status and territory location[END_REF] and the resulting colony instability at that time (Viblanc et al. 2014b) may distract individual birds from surveying for predators. These results are consistent with reports from other species. For instance, in a study that surveyed both a South American cichlid fish, Nannacara anomala, and the willow warbler, Phylloscopus trochilus, male-male conflicts reduced vigilance behavior and resulted in a delay of predator detection when shown the image of a predator [START_REF] Jakobsson | Escalated fighting behaviour incurs increased predation risk[END_REF] A similar result was found in another cichlid species, Neolamprologus pulcher [START_REF] Hess | Territorial aggression reduces vigilance but increases aggression towards predators in a cooperatively breeding fish[END_REF]. Similarly, European robins, Erithacus rubecula, were significantly slower to react to a stuffed sparrowhawk, Accipiter nisus, when engaged in territorial defense [START_REF] Dunn | Trade-offs and seasonal variation in territorial defence and predator evasion in the European Robin Erithacus rubecula[END_REF]. Later in the breeding season (at maximum colony density), king penguins are already settled into their territories and colony density is relatively stable [START_REF] Stier | Starting with a handicap: phenotypic differences between early-and late-born king penguin chicks and their survival correlates[END_REF]. At high density, the information about disturbances may disseminate rapidly from neighbor to neighbor and may aid in searching for and detecting predators (a ripple of disturbance is caused by entering the colony, which alerts neighboring individuals of approaching threats, TLH, VAV, PB pers. obs.) (see also [START_REF] Hernández-Matías | Predation on common tern eggs in relation to subcolony size, nest aggregation and breeding synchrony[END_REF]. Hence, at low colony densities, the distraction hypothesis appears supported, but at high colony densities the many eyes hypothesis is supported. Furthermore, those results may be amplified in concert with predation pressure on the eggs of incubating king penguins which varies across the breeding season. Indeed, previous studies show that predation pressure increases over the breeding season [START_REF] Descamps | Impact of predation on king penguin Aptenodytes patagonicus in Crozet Archipelago[END_REF]. With low predation pressure and low colony density at the start of the breeding season, breeding king penguins may be more attentive to their social environment than to predators. In contrast, with higher predation pressure later in the season when the colony is at its peak density, breeders would shift their attention more towards predators and antipredator responses than to their peers [START_REF] Descamps | Impact of predation on king penguin Aptenodytes patagonicus in Crozet Archipelago[END_REF].

In contrast to AD, bird flight initiation distance (FID) is expected to vary with the "dilution effect", "distracted prey" and "selfish herd" hypotheses. All hypotheses predict similar responses in terms of FID. FID is the result of a decision-making process that occurs after a predator is detected. On one hand, as the number of individuals within a group increases, individual predation risk decreases, and therefore FID should be reduced ("dilution effect" hypothesis). On the other hand, as the number of individuals increases, so does the number of social distractions (e.g., aggression between neighbors), which in turn should distract the focal individuals from performing the necessary decisions needed to initiate flight at an appropriate time, causing FID to be delayed. In the "selfish herd" hypothesis, central positions face reduced predation risk, and the colony is more densely packed, and should therefore have shorter FID.

Yet, FID was not impacted by relative local neighbor density, overall colony density, rank, or aggression with neighbors in incubating king penguins, and therefore neither hypothesis appeared to be strongly supported. One potential explanation to this result may have to do with the cost of reproduction shaping optimal flight initiation distances [START_REF] Cooper | Optimal flight initiation distance[END_REF].

In king penguins, truly fleeing from an approaching predator means abandoning the egg/chick and losing reproduction for the year. Most often, flight is attempted by slowly moving away with the egg or young chick on top of the feetan action that is obviously mechanically constrained.

In addition, even attempting to slowly move away with the egg or young chick may trigger aggressive responses from neighboring conspecifics and increase the risk of injuries and damage to the egg. Indeed, the commitment to reproduction is especially high in king penguins, as it takes the cooperation of a breeding pair for over 14 months to raise a single chick, and successful reproduction occurs at best every two years [START_REF] Jiguet | Individual breeding decisions and long-term reproductive strategy in the King Penguin Aptenodytes patagonicus[END_REF]. Thus, there may be strong selection in this species for behaviors aimed at the early detection of approaching threats and for active territory defense [START_REF] Côté | Aggressiveness in king penguins in relation to reproductive status and territory location[END_REF] despite risks of injury, rather than fleeing altogether from approaching predators, given the elevated fitness costs of abandoning reproduction. FID may then be a relatively inflexible trait in breeding individuals of this species and therefore dilution or distraction effects on this trait may be small. Our previous study found that FID and the decision to flee were significantly repeatable, highlighting moderate to strong individual components in incubating king penguins [START_REF] Hammer | Repeatability of alert and flight initiation distances in king penguins: effects of colony, approach speed, and weather[END_REF]. It is perhaps then not surprising that personality traits were not significantly affected by the social environment, whereas AD, a trait found to be highly flexible and lowly repeatable [START_REF] Hammer | Repeatability of alert and flight initiation distances in king penguins: effects of colony, approach speed, and weather[END_REF], was.

We found that the decision to initiate flight in this species was significantly reduced when the focal bird was engaged in social aggression with neighboring conspecifics, a result which supports the "distracted prey" hypothesis. In this species, territorial defense of incubation and brooding territories is high [START_REF] Côté | Aggressiveness in king penguins in relation to reproductive status and territory location[END_REF], and the "distracted prey" hypothesis suggests that time spent processing social cues and engaging in social interactions with conspecifics may undermine the ability to perform anti-predator behaviors such as vigilance and flight [START_REF] Chan | Anthropogenic noise affects risk assessment and attention: The distracted prey hypothesis[END_REF]). It appears that for king penguins, social aggression may be a strong distraction from the ability to decide when to initiate flight. Although previous studies have not considered how social aggression may affect individual responses to approaching predators, allogrooming impala, Aepyceros melampus, had reduced vigilance rates and responded on average 4 seconds later to a simulated predator than non-allogrooming individuals [START_REF] Mooring | Costs of allogrooming in impala: distraction from vigilance[END_REF].

Anoles, Anolis sagrei, when presented with a conspecific tolerated closer approaches of a model predator before initiating flight [START_REF] Yee | The costs of conspecifics: are social distractions or environmental distractions more salient?[END_REF]. Of particular interest is that territorial defense, such is seen in king penguins, is particularly distracting for focal individuals (as discussed above). For example, in a South American cichlid fish, Nannacara anomala, individuals engaged in territorial mouth wrestling showed reduced vigilance, and chose low intensity behaviors such as display and tail beating when in the presence of a model predator [START_REF] Brick | Fighting behaviour, vigilance and predation risk in the cichlid fish Nannacara anomala[END_REF].

Many studies have focused on prey behavioral responses to predators in the context of foraging [START_REF] Fernández | Effect of group size on individual and collective vigilance in greater rheas[END_REF][START_REF] Randler | Vigilance during preening in coots Fulica atra[END_REF][START_REF] Fairbanks | Mechanisms of the group-size effect on vigilance in Columbian ground squirrels: dilution versus detection[END_REF][START_REF] Rieucau | Many eyes or many ewes: vigilance tactics in female bighorn sheep Ovis canadensis vary according to reproductive status[END_REF][START_REF] Taraborelli | Vigilance and foraging behaviour in a social desert rodent, Microcavia australis (rodentia caviidae)[END_REF][START_REF] Schmitt | Determining the relative importance of dilution and detection for zebra foraging in mixed-species herds[END_REF], urbanization [START_REF] Chapman | Behavioural correlates of urbanisation in the Cape ground squirrel Xerus inauris[END_REF][START_REF] Møller | Urban habitats and feeders both contribute to flight initiation distance reduction in birds[END_REF][START_REF] Hall | Flight initiation distance changes across landscapes and habitats in a successful urban coloniser[END_REF][START_REF] Morelli | Contagious fear: Escape behavior increases with flock size in European gregarious birds[END_REF], distance to refuges [START_REF] Dill | The influence of distance to refuge on flight initiation distance in the gray squirrel (Sciurus carolinensis)[END_REF][START_REF] Bonenfant | The influence of distance to burrow on flight initiation distance in the woodchuck, Marmota monax[END_REF][START_REF] Engelhardt | Effects of levels of human exposure on flight initiation distance and distance to refuge in foraging eastern gray squirrels (Sciurus carolinensis)[END_REF][START_REF] Morelli | Flight initiation distance and refuge in urban birds[END_REF], or in relation to sexual dimorphism [START_REF] Møller | Flight initiation distances in relation to sexual dichromatism and body size in birds from three continents[END_REF][START_REF] Møller | Flight initiation distance, color and camouflage[END_REF][START_REF] Kalb | Flight initiation distance and escape behavior in the black redstart (Phoenicurus ochruros)[END_REF][START_REF] Hensley | Vivid birds do not initiate flight sooner despite their potential conspicuousness[END_REF][START_REF] Samia | Sex differences in lizard escape decisions vary with latitude, but not sexual dimorphism[END_REF].

In contrast, ours focused on the existence of a trade-off between vigilance and flight behavior depending on the individual perceptions of the social and predatory environments in the context of colonial breeding [START_REF] Williams | Natural selection, the costs of reproduction, and a refinement of Lack's principle[END_REF]. Colonial breeding in seabirds has mostly been suggested to emerge as a consequence of the utilization of patchy and unpredictable marine food resources and limited favorable breeding grounds [START_REF] Clode | Colonially breeding seabirds: predators or prey?[END_REF][START_REF] Boulinier | Timing of prospecting and the value of information in a colonial breeding bird[END_REF][START_REF] Danchin | The evolution of coloniality: the emergence of new perspectives[END_REF][START_REF] Boulinier | Public information affects breeding dispersal in a colonial bird: Kittiwakes cue on neighbours[END_REF]. The role predation plays in its evolution, however, remains unclear [START_REF] Rolland | The evolution of coloniality in birds in relation to food, habitat, predation, and life-history traits: a comparative analysis[END_REF]. Several studies have reported antipredator benefits to colonial breeding, by diminishing individual predation risk through dilution, predator-mobbing, or selfish herd effects [START_REF] Brown | Group-living in cliff swallows as an advantage in avoiding predators[END_REF][START_REF] Picman | Antipredation value of colonial nesting in yellow-headed blackbirds[END_REF][START_REF] Hoogland | Advantages and disadvantages of bank swallow (Riparia riparia) coloniality[END_REF]. Our results are consistent with apparent anti-predator benefits of colonial breeding which comes in the form of shared vigilance and predator detectability, but that are traded-off with costs and loss of predator detectability due to social distractions.

Although our present study provided little support for selfish herd effects in king Viblanc et al. 2015) in addition to behavioral responses to approaching predators may yield some valuable information (Viblanc et al. 2015).

To conclude, our study found that the "many-eyes" hypothesis was supported at maximum colony density, while the "distracted prey" hypothesis was supported at lower colony densities in incubating king penguins. In addition, focal birds that were engaged in social aggression with neighboring conspecifics were less likely to initiate flight. We found little support for the "selfish herd" hypothesis based on bird centrality in the colony, and no evidence that the dilution effect was strongly influential. These results show that prey behavioral decisions towards approaching threats are complex, and likely explained by a mix of mutually nonexclusive hypotheses. Future studies on prey reactions to predators should consider testing for joint effects of environmental and social factors in understanding risk-benefit assessments in wild animals.

  individuals were divided into four groups of 50 birds, measured approximately 15 days apart from mid-November 2011 to mid-January 2012. As colony density increases over the breeding season (see Viblanc et al. 2014b for colony dynamics), our objective was to sample birds in natural conditions of increasing social density. Thus, we defined four groups corresponding to periods of increasing overall colony density with: low density (mean  SE birds/m 2 : 4.05  0.11, 19-22 Nov. 2011), medium density (4.48  0.14, 3-5 Dec. 2011), high density (5.04  0.11, 22-23 Dec 2011), and maximal density (5.47  0.11, 13-16 Jan 2012).

  the entire season, individuals (N = 194) became alert on average at7.31  1.91 m; (range = 3.41 -12.3 m). The interaction between relative local neighbor density and overall colony density was significant (LMM, F 3,173.3 = 2.84, P = 0.039) (Fig.2). AD decreased with increasing relative local neighbor density at low (slope = -0.48, 95% CI [-1.02, 0.06]) (Fig.3). However, at medium (slope = -0.46, 95% CI [-95, 0.04]) and high (slope = -0.12, 95%CI [-0.71, 0.47]) overall colony density, there was no relationship between AD and relative local neighbor density. Finally, at maximal colony density AD increased with increasing relative local neighbor density (slope= 0.64, 95% CI [0.02, 1.26]). AD was not significantly affected by the number of aggressive interactions between the focal individual and their neighbors (F 1,172.7 < 1), nor by relative colony rank (F 1,175.0 < 1).

FIGURE CAPTIONS Figure 1 .

 CAPTIONS1 FIGURE CAPTIONS

Figure 2 .

 2 Figure 2. Variables affecting Alert Distance (AD) in incubating king penguins (Aptenodytes

Figure 3 .

 3 Figure 3. The significant effect of the interaction between relative local neighbour density (number of birds/m 2 ) and colony density (low, medium, high, and max) on Alert Distance

Figure 4 .

 4 Figure 4. Variables affected the probability of initiating flight in incubating king penguins

Figure 5 .

 5 Figure 5. Variables affecting Flight Initiation Distance (FID) in incubating king penguins

Fig 2 .

 2 Fig 2.

Fig

  Fig 5.

  penguins, observations in the field show that birds subject to an experimental reduction in local breeding density (by means of exclosure pens) cluster closer together rather than use the available freed-up space (Viblanc 2011), consistent with the selfish herd notion of minimizing individual predation risk. Clustering close to conspecifics combined with the high territorial and aggressive behavior of king penguins[START_REF] Côté | Aggressiveness in king penguins in relation to reproductive status and territory location[END_REF] results in producing a tightly woven network or aggressive birds, making it hard for predators to land in the middle of the colony (TLH, PB,[START_REF] Schmidt | The influence of subcolony-scale nesting habitat on the reproductive success of Adélie penguins[END_REF]. King penguins incubate their single egg on their feet, hence, virtually nothing is known on how closely individuals retain breeding sites year after year, nor whether variation in individual's ability to maintain a breeding site may lead to a situation of entrapment into suboptimal conditions. Future studies should aim at understanding how the "dilution effect", "many-eyes", "selfish herd", and "distracted prey" hypotheses are shaped not only by social and predatory environments, but in interaction with local topography and individual quality. In addition, nothing is known on how such interactions might be shaped by residual reproductive value. For instance, studies in seabirds have found that older parents for which residual reproductive value is low are less responsive to stressors[START_REF] Heidinger | Older parents are less responsive to a stressor in a long-lived seabird: a mechanism for increased reproductive performance with age?[END_REF][START_REF] Heidinger | Mellowing with age: older parents are less responsive to a stressor in a long-lived seabird[END_REF], but see[START_REF] Elliott | The prudent parent meets old age: a high stress response in very old seabirds supports the terminal restraint hypothesis[END_REF]. It would thus be of interest to understand how age and residual reproductive values factor into affecting physiological stress responses in addition to behavior in shaping antipredator decisions.While overall colony density increases as the breeding season progresses, reproductive investment of the breeding individuals also changes with advancing breeding season. Two factors might affect the responses of incubating birds sampled early or late in the breeding season. First, birds that entered reproduction early in the season come nearer to hatching as the breeding season progresses, and hence reproductive investment should increase. It is likely that, In turn, the lower value of reproduction later in the season may render individuals less willing to defend their brood and more likely to flee. Unfortunately, not knowing the incubation stage of birds in our study, we were unable to differentiate responses under those different scenarios. Thus, future studies are needed to specifically test if anti-predator responses are linked to reproductive value and breeding advancement in the king penguin as found in other species[START_REF] Albrecht | Nest crypsis, reproductive value of a clutch and escape decisions in incubating female mallards Anas platyrhynchos[END_REF][START_REF] Osiejuk | Factors affecting flushing distance in incubating female greylag geese Anser anser[END_REF]; see also[START_REF] Dowling | Should I stay, or should I go: Modeling optimal flight initiation distance in nesting birds[END_REF]). While we did not see a change in FID or a change in the probability of initiating flight as the breeding season progressed, there remains a link between reproductive success, brood investment, and risk assessment in later breeding birds that needs to be explored in more depth. For instance, focusing on physiological stress responses (e.g., heart rate known to vary with advancing breeding season;

BG, JPR, RG, VAV; personal observations). Of interest is the idea that breeding aggregations in penguins result from a combination of self-organized dynamics related to predation risk (selfish herd effects) and external forcing factors such as topographically limited possibilities of spatial re-arrangement, for instance due to the retainment of nesting sites year after year (e.g., in Adélie penguins, Pygoscelis adeliae, McDowall and Lynch 2019). This may result in trapping individuals into suboptimal spatial arrangements, with strong edge effects (McDowall and Lynch 2019;

as reproductive investment increases, protecting the investment (i.e., deciding to not flee, or to flee later from an approaching threat) may weigh more heavily on anti-predator behavior

[START_REF] Clark | The risks of parenthood. I. General theory and applications[END_REF]

. Second, birds sampled later in the season may be late breeding birds just starting their incubation. Late breeders in king penguins rarely, if ever, succeed, as chicks are most likely to die before or over winter

[START_REF] Stonehouse | The king penguin Aptenodytes patagonica of South Georgia. Falklands Islands Dependencies Survey[END_REF][START_REF] Weimerskirch | The breeding biology and population dynamics of King Penguins Aptenodytes patagonica on the Crozet Islands[END_REF][START_REF] Stier | Starting with a handicap: phenotypic differences between early-and late-born king penguin chicks and their survival correlates[END_REF]

).

ACKNOWLEDGEMENTS

This work was supported by the French Polar Institute (IPEV) through the IPEV 119 ECONERGY polar program, and by the French National Center for Scientific Research (CNRS).

TLH was supported by a PhD scholarship from the Initiative d'excellence (IdEX), Investissements d'Avenir, Ministère de l'Enseignement Supérieur, de la Recherche et de l'Innovation. We are grateful to the Terres Australes et Antarctiques Françaises (TAAF) for providing logistic support in the field, and to the field personnel and field assistants for providing


Siham Benanane

Nour Nawel Cheikh

El-Houda Bendhina

Didier Villemin

Nour El-Houda Bendhina

Ahcène Boumendjel

New coumarin derivatives accessible in short and eco-friendly conditions with antifungal activity

Keywords: Coumarins; Azacoumarins, Aspergillus niger, Antifungal activity

Introduction

Coumarins are a family of compounds with remarkable therapeutic potential [START_REF] Sharifi-Rad | Natural coumarins: exploring the pharmacological complexity and underlying molecular mechanisms[END_REF]. Being naturally occurring in many plant species, they were and remain the subject of studies for potential therapeutic applications [START_REF] Srikrishna | A review on pharmacological properties of coumarins[END_REF]. Moreover, they are used as scaffolds for the design of drug candidates with broad therapeutic applications, targeting major diseases such as, cancer, and infectious diseases [START_REF] Annunziata | An overview of coumarin as a versatile and readily accessible scaffold with broad-ranging biological activities[END_REF]. The chemical modifications performed on naturally occurring coumarins have mostly dealt with the modification of existing substituents and functions or the introduction of new substituents on the carbon skeleton [START_REF] Stefanachi | Coumarin: a natural, privileged and versatile scaffold for bioactive compounds[END_REF][START_REF] Cheikh | A serendipitous conversion of enaminolactone nitriles with primary amines: a new synthesis of substituted 2-aminopyridine derivatives[END_REF]. The availability of modern and recent synthetic methods have boosted the interest toward coumarin-derived bioactive molecules [START_REF] Farid | Modern metalcatalyzed and organocatalytic methods for synthesis of coumarin derivatives: a review[END_REF]. In this regard, coumarins bearing pyridine rings have attracted the interest of medicinal chemists [START_REF] Medina | Coumarin heterocyclic derivatives : chemical synthesis and biological activity[END_REF]. Very recently, coumarins-pyridine hybrids were reported as multitarget drug candidates toward the management of Alzheimer disease [START_REF] Babaei | Novel coumarin -pyridine hybrids as potent multi-target directed ligands aiming at symptoms of Alzheimer's disease[END_REF][START_REF] Villemin | Solventless convenient synthesis of new cyano-2-aminopyridine derivatives from enaminonitriles[END_REF]. In the same vein, coumarins bearing a fused pyridine ring, namely coumarinopyridines were under the spotlight owing to the prevalence of such motifs in naturally occurring alkaloids [START_REF] Huang | Phochrodines a -d , first naturally occurring new chromenopyridines from mangrove entophytic fungus phomopsis sp[END_REF]. Being structurally very close, chromones bearing pyridine rings have been widely investigated for their therapeutic potential in diverse areas [START_REF] Gaspar | Chromone: A Valid Scaffold in Medicinal Chemistry[END_REF][START_REF] Badal | Cytochrome p450 1 enzyme inhibition and anticancer potential of chromene amides from amyris plumieri[END_REF][START_REF] Reis | Chromone as a privileged scaffold in drug discovery: recent advances[END_REF][START_REF] Silva | Chromones: a promising ring System for new anti-inflammatory rrugs[END_REF][START_REF] Roussel | Chromones bearing amino acid residues: Easily accessible and potent inhibitors of the breast cancer resistance protein ABCG2[END_REF][START_REF] Keri | Synthetic and natural coumarins as potent anticonvulsant agents: A review with structure-activity relationship[END_REF][START_REF] Henry | De novo synthesis of substituted pyridines[END_REF][START_REF] Keri | Chromones as a privileged scaffold in drug discovery: a Review[END_REF][START_REF] Sashidhara | Synthesis and evaluation of new coumarin-pyridine hybrids with promising anti-osteoporotic activities[END_REF][START_REF] Fayed | In vivo and in vitro anti-inflammatory, antipyretic and ulcerogenic activities of pyridone and chromenopyridone derivatives, physicochemical and pharmacokinetic studies[END_REF].

The main objective of this work was to set a simple, short and an eco-friendly method to access coumarin-derived compounds bearing nitrogen atoms. The presence of the nitrogen atom may contribute to the biological activity and pharmacokinetic properties of coumarins [START_REF] Lozinski | An alternative approach to the synthesis of 5H-chromeno[4,3-b]pyridin-5-one system using the cleavage of 5H,9Hpyrano[2[END_REF]. Our focus was made on two coumarins series where one or two nitrogen atoms were introduced. The first is azacoumarins (iminocoumarins) in which one of the coumarin oxygen atoms is replaced with a nitrogen and the second is azacoumarins bearing a fused pyridine ring. For both series, we found the activation with microwave in almost solvent-less conditions offers a new and very convenient method. The conditions and the ease of synthesis prevail high potential of the method for diversity-oriented synthesis (DOS) and generate chemical libraries, very useful within the drug discovery area. With the aim to check the input of the generated compounds in one therapeutic area, we focused on aspergillosis infection.

Aspergillosis is an infection caused by Aspergillus fungus. Generally, aspergillus are not harmful for most people that are in good health. However, those with lung diseases and generally with weak immune system are more subject to infection with Aspergillus and likely to develop health problems, including allergy and lung infections [START_REF] Hoenigl | COVID-19-associated fungal infections[END_REF]. Among Aspergillus, Aspergillus niger (A. niger) is probably the most common species. It is a known contaminant of foods and can cause a disease namely black mold to fruits. It is well documented that A. niger is frequently involved in mycosis of the ear where it causes local inflammation and mycelial growth on cerumen (ear wax) on the skin of the ear canal [START_REF] Martínez | The aspergillus niger major allergen (asp n 3) dna-specific sequence is a reliable marker to identify early fungal contamination and postharvest damage in mangifera indica fruit[END_REF]. The therapeutic management of aspergillosis calls for the use of antifungals such as azoles, polyenes and echinocandines, in mono or combination therapy [START_REF] Yu | Aspergillus niger as a secondary metabolite factory[END_REF]. However, despite the availability of effective drugs against A.niger, the developed resistance toward the treatment in one hand and the emergence of new strains appeals for constant discovery of novel chemical entities with effective antifungal activities [START_REF] Pérez-Cantero | Azole resistance mechanisms in aspergillus: update and recent advances[END_REF]. Recently, it was reported that naturally occurring coumarins and synthetic analogs were of therapeutic potential as anti-aspergillosis [START_REF] Guerra | A new coumarin derivative, 4acetatecoumarin, with antifungal activity and association study against aspergillus spp[END_REF].

Herewith, in the frame of a project aimed to set novel antifungals especially drug candidates targeting resistant A.niger fungus, we studied new coumarin analogs bearing nitrogen atoms.

In this study, we were and particularly interested by azacoumarins (iminocoumarin) and azacoumarinopyridones, easily accessible through novel, short and ecofriendly conditions (Figure 1).

Materials & methods

Chemistry

Follow up of the reactions and checking the homogeneity of the prepared compounds were ensured by using thin-layer chromatography (TLC).Melting points were recorded on a Griffin melting point apparatus and are uncorrected. The reaction under microwave activation was performed using a monomode reactor Synthewave 402 from Prolabo.IR spectra were recorded using KBr disks using Jasco FT-IR-6300 spectrophotometer. 1 H NMR (250 MHz, Bruker AC 250) or (400 MHz, Bruker AC 400 ) and 13 C NMR (62.5 MHz or 150 MHz) spectra were recorded using DMSO-d 6 or CDCl 3 as a solvent with TMS as internal standard. Chemical shifts are reported in parts per million (ppm) and the coupling constants J in Hz. The signals are denoted by the following abbreviations: s: singlet, d: doublet, t: triplet, dd: doublet of doublet, q: quadruplet, m: multiplet. Electrospray ionization (ESI) mass spectra were recorded at the Institut de Chimie Moléculaire de Grenoble (ICMG) on a Waters Xevo G2-S Q TOF instrument (Guyancourt, France) with a nanospray inlet. Liquid Chromatography-Mass Spectrometry (LC-MS) were performed using an LP pmp, a diode-array (DAD) UV detector.

Electospray ionization (ES + and/ES -) was used as ionization method.

General procedure for the preparation of 2-cyano-N-acetamides (3)

In a 50 ml flask fitted were added respectively, a catalytic amount of sodium ethanolate dissolved in 2 mL of ethanol, primary amine (1, 1 eq.) and ethyl cyanoacetate (2, 1 eq.). The reaction mixture was stirred for 6 hours at room temperature. The solid formed was washed several times with diethyl ether and filtered on a sintered glass filter of porosity 0.4 Å to yield compounds (3) as solids.

2-Cyanoacetamide (3a

General procedure for the preparation ofaryl-2-Imino-4-methyl-2H-chromene-3carboxylic acid (aryl)-amide (5).

2-Hydroxyacetophenone (4, 1 eq.) 2-cyano-Nethylacetamide 3a-g (1g, 8.92 mmol, 1 eq) in the presence of two drops of piperidine were heated under microwaves activation (2450 MHz, 120 W) for 1 min. After cooling, the diethyl ether was added and the mixture was stirred for 15 min. The precipitate obtained is directly filtered and then washed several times with diethyl ether and recrystallized with ethanol to yield iminocoumarins (5a-g). The purity of compounds (5a-g) was assessed by NMR and LCMS.

2-Imino-4-methyl-2H-chromene-3-carboxylic acid amide (5a). Mustard yellow

2-Imino-4-methyl-2H-chromene-3-carboxylic acid butylamide (5d).

Green solid; yield: 27%. m.p. 210-212 °C; 1 H NMR (400 MHz, CDCl 3 ) δ 8.04 (s, 1H), 6.98-6.89 (m, 1H), 7.12 (dd, J=8.1 Hz, J=4.5 Hz, 1H), 6.66 (dd, J=6.6 Hz, J=3.0 Hz, 1H), 6.16 (s, 1H), 3.17 (q, J=6.1 Hz, 2H), 1.59 (q, J=6.0 Hz, 2H), 1.35 (q, J=5.9 Hz, 2H), 1.69 (t, J=7.2 Hz, 3H), 0.98 (q, J=6.3 Hz, 3H); 13 General procedure for the preparation coumarinopyridones (9). Enamino-iminocoumarin (7, 1eq) with primary amine 8 (1 eq) were introduced into a 100 mL bicol flask equipped with a condenser and a thermometer. The reaction mixture was heated under reflux of DMF at 90 °C for 2 h or under microwave irradiation for 2 to 7 min. The solution takes on an increasingly dark brown color. The recovered mixture is decanted three times with dichloromethane and washed several times with distilled water in order to remove the DMF.

2-Imino-4-methyl-2H-chromene-3-carboxylic acid (furan-2-ylmethyl)-amide (5f

The crude was purified by chromatography over silica gel using ethyl acetate/cyclohexane (7/3) as an eluent to provide coumarinopyridones (9a-d). The purity of compounds (9a-d) was assessed by NMR and LCMS.

3-Amino-5-imino-3,5-dihydro-chromeno [3,4-c]pyridin-4-one (9a). Brown solid; yield: 66%.

3-Ethyl-5-imino-3,5-dihydro-chromeno[3,4-c]pyridin-4-one (9b

3-Furan-2-ylmethyl-5-imino-3,5-dihydro-chromeno[3,4-c]pyridin-4-one (9c).

3-Benzo[1,3]dioxol-5-ylmethyl-5-imino-3,5-dihydro-chromeno[3,4-c]pyridin-4-one (9d).

Brick and DMF (5 mL) as solvent. The reaction mixture was heated under reflux at 90°C for 6 h.

The brown solution obtained was cooled and washed several times with distilled water and dichloromethane to remove the DMF. The crude was purified over column chromatography eluted ethyl acetate/cyclohexane (3:7) to provide a pure product after recrystallization in ethanol. The purity of compounds (12 and 13) was assessed by NMR and LCMS.

5-Imino-3-[2-(5-imino-4-oxo-5,6-dihydro-4H-benzo[f]isoquinolin-3-yl)-cyclohexyl]-3,5-

dihydro-chromeno [3,4-c] LCMS: R t = 5.22 min, ES+ = 505.2

5-Imino-3-[3-(5-imino-4-oxo-5,6-dihydro-4H-benzo[f]isoquinolin-3-ylmethyl)-benzyl]-3,5-

dihydro-chromeno [3,4-c]

Antifungal activity assessment

Aspergillus niger was chosen for this study, the fungi was obtained from the collection of department of biology of the faculty of science, Béchar University, Algeria. The micro-culture method [START_REF] Wei | Isolation of bioactive compounds, antibacterial activity, and action mechanism of spore powder from Aspergillus niger[END_REF][START_REF] Burketová | The identification of fungi -an illustrated introduction with keys, glossary, and guide to literature[END_REF] was used to confirm the Aspergillus genera. For the species confirmation, single spore method was used to culture A. niger species on three different media: malt extract agar at 25 °C, glycerol nitrate agar (G25N) at 25 °C, and Czapek yeast agar at 5 °C and 37 °C.

Observation was made after 14 days using Pitt and Hocking's identification keys [START_REF] Pitt | Fungi and food spoilage[END_REF].

The compounds at different doses (2, 4, 8 and 10 mg) were poured into petri dishes after being thoroughly shaken under aseptic conditions. After the agars had cooled, the mold was seeded. At a temperature of 25-27°C, the petri dishes were incubated [START_REF] Devecioglu | Antifungal Activities of Different Essential Oils and Their Electrospun Nanofibers against Aspergillus and Penicillium Species Isolated from Bread[END_REF]. The assay was carried out in triplicate with a control sample.

To assess the inhibition of mycelia growth of fungi by hydrosols, the fungi colonies diameters were measured, since the day of incubation and the hydrosol inhibition percentage was calculated using the following formula: I= ((DT-D)/DT)*100, where: DT is mean diameter of mycelia growth in control and D is mean diameter of mycelia growth in treatment [START_REF] Singh | Synthesis and in vitro cytotoxic activity of chromenopyridones[END_REF].

Cytotoxicity

The MTT colorimetric assay was used for the evaluation of the cytotoxicity of the compound 9d. Cells were seeded into 96-well plates at a density of 1×10 5 cells/well for a total growth medium volume of 100 µL and incubated overnight. 100 µL of fresh medium containing compound 9d (dissolved in DMSO in a 0, 1 and 10 μM concentration) were added to each well while DMSO control was fixed at 0.5% (v/v). After 72 h incubation at 37 °C, MTT dye in PBS (5 mg/mL) was added and incubation was prolonged for an additional 4 h. After medium removal and drying, the formazan dye crystals were solubilized in DMSO/ethanol (1:1, v/v, 200 μL). The absorbance was measured at 570 nm and 690 nm as reference wavelength. The effect of 9d on cell viability was calculated as the difference in absorbance between test wells and medium control wells. For both concentrations, the cell viability was higher than 90%.

Results and discussion

The access to the targeted compounds shown in Figure 1 was conducted according to scheme 1 in one step and solvent free conditions starting from N-alkylcyanoacetamids (3) and 2'hydroxyacetophenone (4). In the case where N-alkylcyanoacetamids (3) are not commercially available, we found a novel and rapid method for their synthesis in medium to very good yield (57 to 90%) by the reaction of primary amines (1) with ethyl cyanoacetates (2) using sodium in catalytic amounts in methanol. Herewith, the cyanoacetamides (3) were engaged in a Knoevenagel reaction with hydroxyacetophenone (4) in the presence of piperidine under microwave irradiation to obtain azacoumarins (5) in yields ranging from 27 to 72%. It should highlight that the microwave conditions were highly advantageous over classical conditions (6 hours, 90 °C in DMF). Although, we did not observe a significant difference in yields between the two methods, however, the reaction time is dramatically shorter for the MWassisted method (2 to 7 minutes) and the reaction works in solvent free conditions. For the azacoumarinopyridones series, they were obtained in two steps starting from iminocoumarins (5), according to scheme 2. The iminocoumarins (5) were reacted with dimethylformamide dimethyl acetal (6) under microwave irradiation and solvent-free conditions to afford the corresponding enaminoiminocoumarins [START_REF] Medina | Coumarin heterocyclic derivatives : chemical synthesis and biological activity[END_REF] with 47% yield. The microwave conditions were highly advantageous over classical conditions. The last step is the reaction of enaminoiminocoumarins [START_REF] Medina | Coumarin heterocyclic derivatives : chemical synthesis and biological activity[END_REF] with primary amines (8) by using microwave irradiation without solvent to afford chromenopyridones [START_REF] Villemin | Solventless convenient synthesis of new cyano-2-aminopyridine derivatives from enaminonitriles[END_REF] with yields ranging from 47 to 69%. Here again, the MW irradiation was found to be more effective over conventional heating (DMF at reflux). The structures of synthesized iminocoumarins (5) and coumarinopyridones [START_REF] Villemin | Solventless convenient synthesis of new cyano-2-aminopyridine derivatives from enaminonitriles[END_REF] are shown in Table 1. In order to increase the generality of the synthesis method and to go for further pharmacomodulation, we synthesized two symmetrical diazacoumarinopyridones linked through diamines ( 10) and ( 11) (Scheme 3) to afford derivatives ( 12) and ( 13) with moderate yields (31 and 18%, respectively). This action was taken in the context of multivalent active compounds that are structures bearing at least two identic or different motifs. Becoming frequent within the field of drug discovery area, they are conceived to gain in synergy in terms of biological activity [START_REF] Nudelman | Dimeric drugs[END_REF]. The newly synthesized compounds were tested for in vitro antifungal activity against A. niger, using a growth radial method in agar medium [START_REF] Saleem | A comparison of the radial growth of Aspergillus niger on various culture media prepared by the plant based extracts and potato dextrose agar[END_REF]. The compounds were dissolved in DMSO at different concentrations (2, 4, 8, and 10 mg). The antifungal drug amphotericin B and DMSO were used as a positive and negative control, respectively. To assess the antifungal effect, the diameters of mold mycelail growth were measured in millimeters on the day-7 of incubation, and hydrosol inhibition percentages were calculated using the following formula:

I= ((DT-D)/DT)*100 where DT is mean diameter of mycelial growth in control and D is mean diameter of mycelial growth in treatment [START_REF] Saleem | A comparison of the radial growth of Aspergillus niger on various culture media prepared by the plant based extracts and potato dextrose agar[END_REF].

The obtained results are shown in Table 1.For all tested compounds, the diameters of A. niger colony is reduced following the increase of the concentration of tested compounds with clear relation between the structure and the activity of compounds is observed.

Among the azaocoumarins series (5a-g), the antifungal activity is influenced by the size and the nature of the group linked to the amide function. Indeed, the optimal chain length is an ethyl group (compound 5b versus 5c and 5d). Moreover, the presence of a bulky or an aryl group at the amide function is synonymous of even higher activity as can be observed with compounds (5e-g). The correlation between the size and the shape of the substituent at the amide part can inform about the accommodation of the within the active site of the strain of A. Niger. Regarding the azacoumarinopyridone series (9a-d), the same structureactivity relationship tendency is observed where the presence of an aryl group at the lactam of the pyridine moiety is crucial (compounds 9c and 9d versus 9a and 9b). In order to go forward through the potential development of the most active compound, we evaluated the cellular toxicity of derivative 9d by using the MTT test on the HEK293 (human embryonic kidney cells). Tested at 10 mM, it showed a high cell viability (> 90%), indicating prevailing a high therapeutic index (high biological activity versus low toxicity).

Conclusion

In this work we report a novel, short and eco-friendly synthesis of coumarins derivatives. The method is general and applicable to the synthesis of large chemical libraries. The synthesized compounds can find interest in diverse therapeutic area. Within this work we were interested by the antifungal activity and especially the effect against A.niger. The high antifungal activity and the innocuity of the most active compound are two key requirement for further development. The insertion of one or two nitrogen atoms within the coumarin scaffold offers multiple opportunities for pharmacomodulation. One of the pharmacomodulation point would be the imino group which can be easily reduced to primary amine. The latter can undergo alkylation, acylation and reductive-amination to provide diversity. The later diversity-oriented synthesis is eased by the reported method allows the synthesis of iminocoumarins and coumarinopyridones in large scale.

Future perspectives

Our work report a short and eco-friendly method for the preparation of nitrogen-containing coumaris that could have potential in the medicinal chemistry field. The method allows rapidgeneration of chemical libraries of compounds with functional diversity. The therapeutic potential of synthesized compounds was assessed through the evaluation of their activity as antifungals. The synthetic conditions of our method will be useful for the achievement of diversity oriented synthesis (DOS), a valuable tool for compounds screening in the for drug design field.

Summary points

• Microwave activation was applied as a green method for the synthesis of imino and coumarins and pyridnocoumarins.

• The method was applied to the synthesis of a series of compounds including dimers-like derivatives.

• The antifungal activity against strains of Aspergillus niger was assessed and demonstrated potent effect as compared with a reference drug.

• The reported synthetic method may serve as a rapid and eco-friendly synthesis of nitrogencontaining coumarins for chemical libraries generation.

Financial & competing interests disclosure

This work was supported by CBH-EUR-GS (ANR-17-EURE-0003) of the Université Grenoble Alpes. The authors have no other financial involvement with any organization or entity related to this wrok. The authors declare no conflicts of interest regarding the publication of this manuscript.

No writing assistance was utilized in the production of this manuscript.

Figure 1 .

 1 Figure 1. Structures of some naturally occurring coumarinopyridines (hochrodines) and compounds targeted in this study.

5 (

 5 solid; yield: 56%. m.p. 118-120 °C; 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.01 (s, 2H), 7.54 (d, J= 6,6 Hz 1H), 7.30 (m, J = 6.6 Hz, 1H), 7.25 (dd, J = 8.1 Hz, J = 3.0 Hz,1H), 6.61 (dd, J =8.1 Hz, J =3.0 Hz,1H), 6.16 (s, 1H), 1.03 (s, 3H); 13 C NMR (100.6 MHz, DMSO-d 6 ) δ 175.3 (C), 165.5(C), 162.0 (C), 154.2(C), 152.1 (CH), 134.7 (CH), 125.3 (C), 125.1 (CH), 121.3 (CH), 117.2 (C), 17.8 (CH), IR (KBr) ν (cm -1 ): 3502, 3191, 1650, 1724 ; MS m/z: 203 (M+1). LCMS: R t = 4.12 min, ES+ = 203.22-Imino-4-methyl-2H-chromene-3-carboxylic acid ethylamide (5b). Brown solid; yield: 36%. m.p. 195-197 °C; 1 H NMR (400 MHz, CDCl 3 ) δ 7.57 (dd, J=14.4 Hz, J=3.2 Hz, 1H), 7.26 (dd, J=8.0 Hz, J=6.8 Hz, 1H), 6.15 (s, 1H), 6.16 (s, 1H), 3.23 (d, J=2.8 Hz, 2H), 2,63 (s, 3H), 1,6 (t, 3H); 13 C NMR (100.6 MHz, CDCl 3 ) δ 175.3 (C), 165.5 (C), 162.04 (C), 154.2 (C), 152.1 (CH), 134.79 (CH), 125.6 (C), 125.0 (CH), 121.3 (CH), 117.2 (C), 36.9 (CH 2 ), 18.CH 3 ), 16.8 (CH 3 ); IR (KBr) ν (cm -1 ): 3295, 2260, 1661, 1554, 1724. MS m/z: 231 (M+H).LCMS: R t = 3.98 min, ES+ = 231.5 2-Imino-4-methyl-2H-chromene-3-carboxylic acid propylamide (5c). Brown solid. yield: 31%. m.p. 200-202 °C; 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.04 (s, 1H), 7.97 (d, J=8.0 Hz, 1H), 7.78 (d, J=7.6 Hz, 1H), 7.47 (dd, J=8.0 Hz, J=3.0 Hz, 1H), 6.16 (s, 1H), 3.56 (s, 1H), 3.04 (t, J=7.2 Hz, 2H), 2.71 (s, 3H), 1.39 (q, 2H), 0.83 (t, 3H); 13 C NMR (100.6 MHz, DMSO-d 6 ) δ 166.5 (C), 164.5 (C), 155.2 (C), 152.1 (C), 127.5 (CH), 126.5 (CH), 122.3 (C), 125.17 (CH), 121.3 (CH), 114.7 (C), 45.1 (CH 2 ), 24.2 (CH 2 ), 18.0 (CH 3 ), 11.4 (CH 3 ); IR (KBr) ν (cm -1 ): 3542, 3297, 2971, 1675, 1721, 1075. MS m/z: 245 (M+H). LCMS: R t = 3.81 min, ES+ = 245.1

  C NMR (100.6 MHz, CDCl 3 ) δ 166.5 (C), 164.5(C), 155.2 (C), 152.1 (C), 127.5 (CH), 126.5 (CH), 122.3 (C), 125.17 (CH), 121.3 (CH), 114.7 (C), 43.1 (CH 2 ), 33.4 (CH 2 ), 20.2 (CH 2 ), 18.01 (CH 3 ), 13.4 (CH 3 ); IR (KBr) ν (cm -1 ): 3375, 3291, 2197, 2998, 1675, 1563; MS m/z: 259 (M+1). LCMS: R t = 3.77 min, ES+ = 259.7 2-Imino-4-methyl-2H-chromene-3-carboxylic acid cyclohexylamide (5e). Orange-yellow solid; yield: 53%. m.p. 208-210 °C; 1 HNMR (400 MHz, CDCl 3 ) δ 7.58 (dd, J=2.1 Hz, J=7.6 Hz, 1H), 7.25 (dd, J=2.1 Hz, J=7.6 Hz, 1H), 6.16 (s, 1H), 3.49 (m, 1H), 3.42 (q, J=4.0 Hz, 2H), 3.25 (q, J=4.0 Hz, 2H), 3.26 (m, 2H), 1.50 (s, 3H); 13 C NMR (100.6 MHz, CDCl 3 ) δ 165.5 (C), 164.5 (C), 154.2 (C), 152.1 (C), 135.2 (CH), 129.1 (CH), 128.5 (C), 126.0 (CH), 125.5 (CH), 111.7 (C), 46.9 (CH), 41.5 (CH 2 ), 41.5 (CH 2 ), 26.2 (CH 2 ), 22.3 (CH 2 ), 22.3 (CH 2 ), 18.3 (CH 3 ); IR (KBr) ν (cm -1 ): 3405, 2269, 1675, 1724; MS m/z: 285 (M+1). LCMS: R t = 4.02 min, ES+ = 285.3

9 (

 9 ; 13 C NMR (100.6 MHz, DMSO-d 6 ) δ 165.5 (C), 162.1 (C), 155.0 (C), 151.2 (C), 138.5 (C), 130.7 (CH), 128.1 (CH), 128.3(CH), 127.3 (CH), 125.3 (CH), 125.2 (CH), 122.5 (C), 121.7 (C), 117.2 (CH), 115.3 (CH), 112.1 (C), 48.5 (CH 2 ), 24.8 (CH 3 ); IR (KBr) ν (cm -1 ): 3642, 3291,1675, 1724; MS m/z: 293 (M+1). LCMS: R t = 3.21 min, ES+ = 293.1 General Procedure for the Preparation of aryl-2-ylmethyl-aryl-3H-chromeno[3,4c]pyridine-4,5-dione from imino-coumarins (7). The azacoumarin (5, 1eq) and dimethyl formamide dimethyl actetal (6, 1eq) were introduced into a 25 ml flask equipped with a magnetic stirrer. The mixture is stirred at room temperature and without solvent for 24 hours or under microwave irradiation for 2 to 7 min. The solution becomes darker. The obtained solid was washed several times with diethyl ether and a few drops of ethanol then proceed to purification with chromatography column. The purified compounds were recrystallized with ethanol.4-(2-Dimethylamino-vinyl)-2-imino-2H-chromene-3-carboxylic acid amide (7).Solid brown, yield: 47%. m.p. 153-155 °C; 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.50 (d, J=5.6 Hz, 1H), 8.19 (d, J=7.2 Hz, 1H), 7.97 (dd, J=7.6 Hz, 1H), 7.69 (d, J=7.6Hz, J=2.5 Hz, 1H), 7.49 (d, J=7.6 Hz, 1H), 6.92 (s, 2H), 5.75 (d, J=5.6 Hz, 1H), 5.74 (d, J=5.6 Hz, 1H), 3.14(s, 6H); 13 C NMR (100.6 MHz, DMSO-d 6 ) δ 168.3 (C), 165.5 (C), 155.0 (C), 155.18 (C), 128.7 (CH), 127.CH), 125.3 (CH), 122.3 (C), 121.3 (CH), 113.2 (CH), 115.2 (C), 104.5 (CH), 42.5 (CH), 42.5 (2x CH 3 ); IR (KBr) ν (cm -1 ): 3291, 2200, 1719, 1675, 1613, 1250. MS m/z: 258 (M+1).

  m.p. 204-206 °C; 1 H NMR (400 MHz, DMSO-d 6 ) δ 7.26 (d, J=4.5 Hz, 1H), 7.32-6,19 (m, 4H), 7.19 (s, 1H), 5.97 (d, J=4.8 Hz, 1H), 4.47 (s, 2H); 13 C NMR (100.6 MHz, DMSO-d 6 ,) δ 167.4 (C), 160.5 (C), 156.3 (C), 155.0 (C), 127.6 (CH), 122.3 (CH), 121.0 (CH), 120.2 (C), 119.3 (CH), 113.3 (CH), 116.2 (CH), 109.3 (C); IR (KBr) ν (cm -1 ): 3316, 2240, 1651, 1603, 1542, 1250. MS m/z: 228 (M+H).

  Brown solid; yield: 47%. m.p. 215-217 °C; 1 H NMR (400 MHz, CDCl 3 ) δ 9.11 (d, J=8.4 Hz, 1H), 8.75 (dd, J=6.2 Hz, J=4.1 Hz, 1H), 7.67 (dd, J=12.0 Hz, J=4.2 Hz, 1H), 7.54 (dd, J=8.1 Hz, J=3.2 Hz, 1H), 7.42 (d, J=4.1 Hz, 1H), 6.73 (d, J=4.1 Hz, 1H), 6.63 (t, J=5.8 Hz, 1H); 6.40 (dd, J=4.8 Hz, J=1.6 Hz, 1H), 4.53 (s, 1H), 4.49 (d, J=10.6 Hz, 1H), 3.39 (d, J=5.6 Hz, 2H), 1.69-1.35 (m, 2H), 1.08-1.04 (m, 2H); 13 C NMR (100.6 MHz, CDCl 3 ) δ 165.5 (C), 159.0 (C), 155.0 (C), 152.1 (C), 146.2 (CH), 129.7 (CH), 127.3 (CH), 124.2 (CH), 122.6 (CH), 119.3 (C), 116.2 (CH), 113.3 (C), 111.2 (CH), 110.1 (CH), 104.3 (CH), 48.0 (CH 2 ); IR (KBr) ν (cm -1 ): 3507, 2932, 1601, 1505, 1349, 1028; MS m/z: 310 (M+18). LCMS: R t = 1.98 min, ES+ = 293.1

Scheme 1 .

 1 Scheme 1. (a) Na, CH 3 OH, room temp.; (b) piperidine, MW, 2 to 7 min.

Scheme 2 .

 2 Scheme 2. (a) MW, 2 to 7 min; (b) MW, 2 to 7 min, or DMF, 90 to 120 °C.

Scheme 3 .

 3 Scheme 3.(a) MW, 2 to 7 min, or DMF, 90 to 120 °C.

propylacetamide (3c). White solid

  NMR (400 MHz, DMSO-d 6 ) δ 8.86 (s, 1H), 7.60 (d, J=4.0 Hz, 1H), 6.45 (d, J= 4.0 Hz, 1H), 6.28 (d, J=4.0 Hz, 1H), 4.29 (d, J=4.0 Hz, 2H), 3.69 (s, 2H); 13 C NMR (100.6 MHz, DMSO-d 6 ) δ 162.58 (C), 151.88 (C), 142.83 (C), 116,61 (C), 110.96 (C), 107.78 (C), 36.35 (CH 2 ), 25.65 (CH 2 ); IR (KBr) ν (cm -1 ): 3276, 2261, 1646, 1351 cm -1 .N-Benzyl-2-cyanoacetamide (3g). White solid; yield 90%. m.p. 119-121 °C; 1 H NMR (250 MHz, CDCl 3 ) δ 8.81 (t, J=4.9 Hz, 1H ), 7.27-7.41 (m, 5H), 4.35 (d, J=5.8 Hz, 2H), 3.76 (s, 2H); 13 C NMR (100.6 MHz, CDCl 3 ) δ 162.15 (CO), 116.15 (CN), 138.5 (5×C arom ) 42.67 (CH 2 Ph), 25.28 (CH 2 CN); MS. m/z: 167 (M+H); IR (KBr) ν(cm -1 ): 3302, 2255, 1644, 1626; HRMS (ES-QTOF) Calcd for C 10 H 11 N 2 O (M+H) 175.0871. Found 175.0873.

	Hz, 6H), 1.43 (q, J=5.8 Hz, 4H); 13 C NMR (100.6 MHz, DMSO-d 6 ) δ 160.87 (C), 116.16 (C),
	48.38 (CH 2 ), 32.03 (CH 2 ), 28.12 (CH 2 ), 25.03 (CH 2 ), 24.84 (CH 2 ), 24.27 (CH 2 ), 24.27 (CH 2 );
	IR (KBr) ν (cm -1 ): 3276, 2261, 1646. EIMS m/z (% relative abundance): 167 (M+H, 52), 85
	(100), 83 (56). HRMS (ES-QTOF) Calcd. for C 9 H 15 N 2 O (M+H) 167.1184. Found 167.1186.
	2-Cyano-N-(furan-3-ylmethyl)acetamide (3f). Orange-yellow solid; yield 85%. m.p. 132-134
	°C; 1 H
	). White solid, yield 85%, m.p. 118-120 °C; 1 H NMR (400 MHz,
	DMSO-d 6 ) δ: 6.06 (s, 1H), 3.39 (s, 2H); 13 C NMR (100.6 MHz, DMSO-d 6 ) δ: 172.19 (C),
	115.05 (C), 25.72 (CH 2 ); IR (KBr) ν (cm -1 ): 3276, 2261, 1646, 1351.
	2-Cyano-N-ethylacetamide (3b).White solid; yield 57%; m.p. 120-122 °C; 1 H NMR(400 MHz,
	DMSO-d 6 ) δ 8.26 (s,1H), 3.59 (s, 2H), 3.11 (q, J=8.0 Hz, 2H), 1.19 (t, J=8.0 Hz, 3H); 13 C
	NMR (100.6 MHz, DMSO-d 6 ) δ: 162.19 (C), 116.75 (C), 34.45 (CH 2 ), 25.72 (CH 2 ), 14.84
	(CH 3 ); IR (KBr) ν (cm -1 ): 3276, 2261, 1646, 1351.
	2-Cyano-N-, yield 67%; m.p. 120-122 °C; 1 H NMR (400
	MHz, DMSO-d 6 ) δ: 8.81 (s, 1H), 3.76 (s, 2H) , 3.31 (t, J=8.0 Hz, 2H), 1.32 (m, 2H) , 0.98 (t,
	J=8.8 Hz, 3H); 13 C NMR (100.6 MHz, DMSO-d 6 ) δ: 162.15 (C), 116.15 (C), 42.67 (CH),
	22.28 (CH), 20.61 (CH 2 ), 14.08 (CH 3 ); IR (KBr) ν (cm -1 ) : 3302, 2255, 1644, 1626.
	N-Butyl-2-cyanoacetamide (3d). White solid; yield 59%. m.p. 100-102 °C; 1 H NMR (400
	MHz, DMSO-d 6 ) δ 8.20 (s, 1H), 3.30 (s, 2H), 3.10 (t, J=5.6 Hz, 2H), 1.4 1 (m, 2H), 1.31 (m,
	2H), 0.90 (t, J=5.6 Hz, 3H); 13 C NMR (100.6 MHz, DMSO-d 6 ) δ 162.32 (C), 116.75 (C),
	39.17 (CH 2 ), 31.33 (CH 2 ), 25.70 (CH 2 ), 19.91 (CH 2 ), 14.03 (CH 3 ); IR (KBr) ν (cm -1 ): 3276,
	2263, 1640, 1349.
	2-Cyano-N-(cyclohexylmethyl)acetamide (3e). White solid; yield 76%. m.p. 122-124 °C; 1 H
	NMR (400 MHz, DMSO-d 6 ) δ 8.13 (s, 1H), 3.58 (s, 2H), 2.01 (m, 10H), 1.76 (quin, J=5.8

  Hz, 2H), 1.45 (s, 3H);13 C NMR (100.6 MHz, CDCl 3 ) δ 166.05 (C), 164.2 (C), 156.4 (C), 152.1 (C), 151.4 (C), 143.9 (CH), 129.2 (CH), 127.1 (CH), 122.2 (C), 121.5 (CH), 115.4 (CH), 112.1 (C), 109.7 (CH), 102.1 (CH), 46.5 (CH 2 ), 19.0 (CH 3 ); IR (KBr) ν (cm -1 ): 3574, 3291,2257, 1675, 1724; MS m/z: 283 (M+1). Hz, 1H), 7.66 (dd, J=6.7 Hz, J=2.2 Hz, 1H), 7.32 (dd, J=6.9 Hz, J=2.1Hz, 4H), 6.61 (dd, J=6.6 Hz, J=3.0 Hz, 1H), 7.41 (m, 1H), 6.16 (s, 1H), 4.84 (d, J=4.8 Hz, 2H), 1.03 (s, 3H)

	LCMS: R t = 2.96 min, ES+ = 283.6
	2-Imino-4-methyl-2H-chromene-3-carboxylic acid benzylamide (5g). Light yellow solid;
	yield: 72%. m.p. 129-131 °C; 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.75 (s, 1H), 8.02 (d, J=6.8
	). Orange-
	yellow solid; yield: 37%. m.p. 150-152 °C; 1 H NMR (400 M Hz, CDCl 3 ) δ 9.89 (large s, 1H),

7.63 (d, J=8.0 Hz, 1H), 7.58 (d, J=7.2 Hz, 1H), 7.31(d, J=9.6 Hz, J=2.0 Hz, 1H), 7.22 (d, J=9.6 Hz, J=2.0 Hz, 1H), 7.13 (d, J=8.0Hz, 1H), 6.26 (s, 1H), 6.21 (s, 1H), 4.37 ( d, J=5.6

Table 1 .

 1 Antifungal activity of iminocoumarins and coumarinopyridones

	Cmpd.	Structure	Compound concentration (mmol)
			Growth inhibition (%)
		9.7	19.8	39.6	49.4
	(5a)	26.2±2.2	40.5±2.9	70.0±1.4	88.6±3.7
		8.5	17.4	34.7	43.4
	(5b)	79.0±2.2	87.1±0.01	94.7±1.64	100±0.01
		8.03	16.4	32.7	40.9
	(5c)	20.0±1.42	40.0±2.8	45.7±0.08	60.0±2.8
		7.6	15.5	31.0	38.7
	(5d)	0±0.00	15.2±8.7	36.6±3.6	50.5±2.2
		6.9	14.0	28.1	35.2
	(5e)	40.0±0.01	73.8±3.0	93.3±0.82	100±0.01
		6.9	14.2	28.3	35.4
	(5f)	69.5±2.2	82.4±3.6	95.7±0.01	100±0.0
		6.7	13.7	27.4	34.2
	(5g)	15.2±1.6	89.5±3.6	100±0.01	100±0.01