domain: sdv info info.info-ts

text:
Yann Doh
email: yanndoh.m2@gmail.com

Fabienne Delfour

Elodie Augier

Hervé Glotin

Christian Graff

Olivier Adam

Elodie Fabienne Augier

Herv{\ ' Glotin

E}

Bottlenose dolphin (Tursiops truncatus) sonar slacks off before touching a non-alimentary target

Keywords: title={Bottlenose dolphin (Tursiops truncatus) sonar slacks Bottlenose dolphin, echolocation, approach phase, terminal buzz, inter-pulse interval, slackening-off

Odontocetes modulate the rhythm of their echolocation clicks to draw information about their environment. When they approach preys to capture, they speed up their emissions to increase the sampling rate of "distant touch" and improve information update. This global acceleration turns into a "terminal buzz" also described in bats, which is a click train with drastic increase in rate, just as reaching the prey. This study documents and analyses under human care bottlenose dolphins' echolocation activity, when approaching non-alimentary targets. Four dolphins' locomotor and clicking behaviours were recorded during training sessions, when sent to immersed objects pointed by their trainers. Results illustrate that these dolphins profusely use echolocation towards immersed non-alimentary objects. They accelerate click emission when approaching the target, thus displaying a classical terminal buzz. However, their terminal buzz slackens off within a quarter of second before the end of click train. Typically, they decelerate to stop clicking just before they touch the object using their rostrum lower tip. They do not emit clicks as the contact lasts. In conclusion, when exploring inert objects, bottlenose dolphins under human accelerate clicking like other odontocetes or bats approaching preys. Bottlenose dolphins' particular slackening-off profile at the end of the buzz shows that they anticipate the moment of direct contact, and they stop just as real touch relays distant touch of the object.

Introduction

Toothed whales such as dolphins, porpoises and sperm whales are equipped with bio-sonar analogous to that of bats [START_REF] Griffin | Echolocation by blind men, bats and radar[END_REF], allowing navigation and detection (Thomas et al., 2003). They echolocate with broadband pulses called clicks [START_REF] Herman | The communications systems of cetaceans[END_REF][START_REF] Au | Hearing by whales and dolphins[END_REF], that are repeatedly emitted in trains. Clicks are short transitory signals: 50 to 80 μs for bottlenose dolphins (Tursiops truncatus) [START_REF] Mann | Detection of ultrasonic tones and simulated dolphin echolocation clicks by a teleost fish, the American shad (Alosa sapidissima)[END_REF]. The duration between two adjacent clicks or inter click interval (ICI) is variable within a train as well as between two trains and ranges from 0.8 ms to 5 ms for a buzz (Luis et al., 2016). Click emission is not automatic but controlled (Moore and Pawloski, 1990). The animal modulates amplitude, frequency content and rhythm of the clicks depending on the echolocating function (e.g., navigation, target detection or discrimination, prey pursuit, social contact).

Modulation of echolocation rhythm during foraging and prey capture has been first described in bats hunting mosquito using ultrasonic pulses comparable to clicks into three phases: search, approach, and terminal, associated to characteristic acoustic behaviours [START_REF] Griffin | Listening in the dark: the acoustic orientation of bats and men[END_REF][START_REF] Griffin | The echolocation of flying insects by bats[END_REF]. Search phase consists in a navigation by the predator in the environment to detect and localize potential preys. It involves ICIs from about 50 to 100 ms. The second phase coined prey approach begins when a prey is detected, approximately at 2-4 m [START_REF] Kalko | Insect pursuit, prey capture and echolocation in pipistrelle bats (microchiroptera)[END_REF]: the animal moves toward the target and pursues it if necessary. It is associated with an acceleration in emission rate, with ICIs about 50 to 10 ms. Finally, the terminal phase, when the predator is close enough to attempt a catch, is associated with a "terminal buzz", comprised of ICIs about 4 to 7 ms. The "buzz" corresponds to the sound heard from an ultrasonic converter, when successive pulses are no more perceived as distinct clicks but when the accelerated train forms a continuous humming.

This phenomenon is found by convergent evolution in other echolocating species, such as sperm whales Physeter macrocephalus [START_REF] Miller | Sperm whale behaviour indicates the use of echolocation click buzzes "creaks" in prey capture[END_REF], beaked whales Ziphidae [START_REF] Madsen | Biosonar performance of foraging beaked whales (Mesoplodon densirostris)[END_REF] and short finned pilot whales (Globicephala macrorhynchus) (Aguilar et al. 2008) and beluga whales (Delphinapterus leucas) [START_REF] Ridgway | Forward shift of feeding buzz components of dolphins and belugas during associative learning reveals a likely connection to reward expectation, pleasure and brain dopamine activation[END_REF] recorded from sound and position sensors directly fixed on the animals. During the search phase, they emit spaced and regular clicks (Mullins et al., 1988;[START_REF] Whitehead | Patterns of visually observable behaviour and vocalizations in groups of female sperm whales[END_REF]. Trains turn to buzzes or "creaks" during capture events attempts that are inferred from accelerations and rapid body movements recorded on the animal [START_REF] Mohl | The monopulsed nature of sperm whale clicks[END_REF][START_REF] Zimmer | Three-dimensional beam pattern of regular sperm whale clicks confirms benthorn hypothesis[END_REF][START_REF] Laplanche | Measuring the off-axis angle and the rotational movements of phonating sperm whales using a single hydrophone[END_REF]. In captivity, few studies on echolocation during prey capture have been documented in odontocetes [START_REF] Verfuss | Echolocation by two foraging harbour porpoises (Phocoena phocoena)[END_REF][START_REF] Miller | Prey capture by Harbor porpoises (Phocoena phocoena): a comparison between echolocators in the field and in captivity[END_REF][START_REF] Wisniewska | Buzzing during biosonar-based interceptiobn of prey in the delphinids Tursiops truncatus and Pseudorca crassidens[END_REF][START_REF] Ridgway | On doing two things at once: dolphin brain and nose coordinate sonar clicks, buzzes and emotional squeals with social sounds during fish capture[END_REF]. Synchronized underwater video -and highfrequency sound -recording showed a speed-up in the animals' clicks when they approach the prey and a decrease when it is at less than 1 m. When dolphins succeed in capturing a prey, they could emit a "victory squeal" 0.2 to 20 s after capture [START_REF] Ridgway | On doing two things at once: dolphin brain and nose coordinate sonar clicks, buzzes and emotional squeals with social sounds during fish capture[END_REF][START_REF] Dibble | Dolphins signal success by producing a victory squeal[END_REF].

As more pulses are emitted for a same duration, the terminal buzz represents a higher sampling rate in time, correlated through movement with a finer resolution in space. The subsequent faster update of information [START_REF] Britton | Echolocation behaviour and prey-capture success in foraging bats: laboratory and field experiments on Myotis Daubentonii[END_REF] responds to the need of stronger temporal and spatial resolution in order to reach a small moving target [START_REF] Madsen | Biosonar performance of foraging beaked whales (Mesoplodon densirostris)[END_REF][START_REF] Verfuss | Echolocation by two foraging harbour porpoises (Phocoena phocoena)[END_REF]. Similarly, needs in electro-location explain why night-active weakly-electric fish increase their rate of discharge pulses when swimming and when facing a novel object [START_REF] Bauer | Electric organ discharge activity of resting and stimulated Gnathonemus petersii (Mormyridae)[END_REF][START_REF] Avril | Active electrolocation of polarized objects by pulse-discharging electric fish, Gnathonemus petersii[END_REF].

The present study investigates the echolocation course in bottlenose dolphin (Tursiops truncatus), during the presentation of static non-alimentary objects. It is based on an ICI duration analysis inspired from electric fish work [START_REF] Graff | Firing activity of the weakly-electric fish Marcusenius macrolepidotus (Mormyridae, Teleostei). Logarithmic distribution of inter-pulse intervals and sequential inequality testing[END_REF]. Unlike most investigations in captive dolphins (e.g. [START_REF] Harley | Bottlenose dolphins perceive object features through echolocation[END_REF], this experiment attempts to explore the echolocation behaviour displayed without performance constraint. Dolphins were not blindfolded and no perceptive task was imposed on the animals. Their use of echolocation was spontaneous, yet they were led to objects. For a better knowledge of the context of their emission, acoustic signals were recorded simultaneously with underwater locomotor behaviour. To control for contextual variables, subjects were individually called and driven by their trainers to the presence of single inert objects. This limited the circumstances of recording and focused on standardized conditions of free interaction. The animals were thus reinforced coming close -not for touching -the objects, and not for echolocating because the trainers were deaf to the clicks.

The experiment documents echolocation in captive T. truncatus, when vision is available in their familiar pools and in normal daily context. Second, as good quality click trains were abundantly recorded, their rhythm was investigated to show their reaction to the pointing to objects immersed near them, with relation to their behaviour. Third, as dolphins eventually touched an object with their rostrum when coming closer, characteristic increases in emission rate with terminal buzz were quantified, comparable to that already described in the approach and terminal phase of prey capture in bats and other odontocetes.

Method

Subjects and housing

Observations focused on four bottlenose dolphins (T. truncatus) housed at Parc Astérix dolphinarium (France) at daytime, during seven regular training sessions, in February 2012. Animals were two females (Beauty and Baily) and two males (Guama and Balasi), respectively 40, 13, 30, and 8 years old. The two older dolphins were born in the wild while the two younger ones were born in Parc Astérix. Animals move and interact at will with six other conspecifics in three interconnected pools, two indoors and one outdoors. About the behavior of captive dolphins, regular published ethograms show their diversity and closeness to the ones of wild conspecifics.

Using a known hand gesture, the trainers commanded the four tested dolphins to swim alone to the recording area. The experiment took place in the largest pool (60m * 28 m, maximal depth 4.5 m) situated outdoors, comprising two advanced platforms facilitating contact with trainers and an underwater window gallery facilitating video recording.

Experimental context

The experiment consisted in simultaneously recording acoustical signals and behaviour of individual dolphins with relation to underwater presentation of initially unfamiliar objects. Objects were harmless and non-eatable: a holed white PVC disc, a holed white wooden disc, a red plastic fin and a plastic bottle with red colored content (Fig. 1). These objects did not emit any kind of acoustical or electrical signals. Since dolphins showed interest for electronic objects [START_REF] Goodson | Interpreting the acoustic pulse emissions of a wild bottlenose dolphin (Tursiops truncatus)[END_REF], the silent omni-directional hydrophone was attached to the object to form an objecthydrophone device. Moreover, clicks are produced ahead in a directive beam in-axis of the animal [START_REF] Au | The acoustic field on the forehead of echolocating Atlantic bottlenose dolphins (Tursiops truncatus)[END_REF]: the objecthydrophone association prevented from signal attenuation occurring when acquisition takes place out of this beam (Nowacek, 1999).

The experiment was conducted during daily training sessions. Sessions took place five times a day and lasted for 20 min. The ten dolphins were called in front of the outdoor beach (Fig. 2), working with their trainer. Since dolphins understand target-pointing (Pack andHerman, 2004, 2007), trainers sent individually off each the four participant dolphins by a hand gesture at the opposite side of the pool (as in [START_REF] Verfuss | Echolocation by two foraging harbour porpoises (Phocoena phocoena)[END_REF] close to the objects. Seclusion prevented as much as possible to record a mix of sounds emitted by different dolphins, without having to physically isolate the individual from its social group. When the focus dolphin arrived close to the experimental platform, a reward (whistle, food and/or vocal encouragement) was given by a second trainer. The experimenter then immersed one object-hydrophone device vertically along the pool wall, about 30 cm below the surface, for approximately 20 to 30 s of presentation in front of the window facing the camera. From above the surface, the trainer pointed the index finger to the object under water. Dolphins were repeatedly submitted to object #1 and to an alternative object. Each session, object #1 was presented first, and more often (M+/-SD = 6.6 +/-1.4 times) than the second one (3.6 +/-1.0 times). For three subjects, the alternative object, either object #2 or #3, was changed between the two sessions. Balasi participated in one session only, using alternative object #4 because of previous encounter with the two other ones. It often happened that several dolphins responded and arrived together to the presenting spot. Their unexpected spontaneous individual and social behaviours precluded better control of encounters, as would have been required to test for specific effects of objects or object change.

Dolphins were free to station, to explore the device or to swim away. We avoided all constraint to the animals. When the trial ended, the device was removed, the animal was sent away and another dolphin could come for a new trial. This procedure was repeated as often as possible, to obtain a maximum of trials for each dolphin and each presented object. In the end, two trials for the same individual in the same session were separated by about 5 min.

Recordings

Each training session was filmed through the underwater viewing gallery, under the experiment platform, by a fixed camera (Panasonic Lumix DMC-FZ38, frame rate: 33 fps) to obtain an accurate behavioral observation while the dolphin emitted sounds and interacted with the object. A hydrophone (Cetacean Research C54XRS) attached to the objects and connected to an analog-to-digital converter (Tascam HD-P2) with an integrated adjustable preamplifier allowed sound signal acquisition. Preliminary tests allowed to adjust the gain to 5dB and keep it set for the whole experiment. The converter saved the sampled signal in *.WAV format on a SD card at 44.1 kHz sampling rate and coded on 16 bits. These recording parameters ensured the task of acoustic events detection for ICI estimates. The expected values are mostly higher than 1 ms (Luis et al., 2016). However, 44.1 kHz sampling is a limiting factor for clicks spectral analysis, as bottlenose dolphin's peak frequency is over the resulting Nyquist frequency. Some clicks were also audible on the camera soundtrack, facilitating audio-video matching of locomotor and acoustic behaviour. Total duration of cumulated sequences of video and audio recording reached about 100 minutes over two days.

Behavioral observations

We distinguished two main behavioral contexts around echolocation: "exploration" defined as a distant scan of the object, not ending with any physical contact and "approach" that occurred when the dolphin moved toward the object-hydrophone device and touched it, in a brief or lasting contact [START_REF] Atem | Changes in click source levels with distance to targets: studies of free-ranging white-beaked dolphins Lagenorhynchus albirostris and captive harbour porpoise Phocoena phocoena[END_REF]Ve rfuss et al., 2009). As the trainer pointed to the device, dolphins eventually positioned against it with their rostrum. We called this behaviour "positioning". During a long static positioning on the device a dolphin could briefly loose the contact because of the object moving with wavelets. The animals approached again, we called these low amplitude movements repositioning considered as minor adjustments.

Behavioral analysis

A total of 74 approaches were satisfactorily filmed with an exploitable soundtrack. Qualitative sound description of recorded approaches revealed if the dolphin clicked while approaching, if it stopped clicking at contact onset, and if it restarted clicking while the contact lasted.

For a further extensive quantitative analysis of ICIs (as in [START_REF] Verfuss | Echolocation by two foraging harbour porpoises (Phocoena phocoena)[END_REF], thirty-eight of them were conservatively excluded, as soon as signal-to-noise ratio was too low or several dolphins clicked simultaneously at the object.

A consistent amount of approaches was selected and 36 good-quality trains (48 % of the complete dataset) were further analyzed into details. The observed acoustic behaviour is thus present at least for ½ approaches which is thus confidently representative of a repetitive acoustic behavior within this dolphin group. When possible, the accurate instant of contact with the device was noted; this was the case in all trials where the dolphin touched a part of the hydrophone (cable or tip) because physical contact is then audible on the recording. When the very moment of contact was impossible to determine, because it was not audible in the sound file, we used the video record to propose a range where it had to be contained. Such ranges remained inaccurate when the rostrum contact was hidden behind the object and because of the limited time precision of the slow-motion capacities of our video hardware (33 fps).

ICI processing

The raw signals in WAV format were preprocessed using the free audio software Audacity (v1.2.3, SourceForge.net). By using high-pass filtering and adjusting the gain, temporal adjustment of the sequences of interest was made and further prepared the click amplitude level above the background noise The ICI time course of the sequence was then extracted with computer assistance.

Using a custom-built detection program written in Matlab, clicks were detected by a trigger based on amplitude (absolute values), then selected and characterized with additional comparison parameters according to biologicallyrelevant criteria.

The detection of standing out amplitude events was operated using first a 34 ms sliding window. In such delay, the amplitude was not likely to be affected by changes in distance and orientation of the source to the hydrophone. Second, the algorithm selected clusters of neighboring samples exceeding the mean by 2.5 to 4.5 times the standard deviation, as peaks in putative clicks. The position of the peak was estimated by selecting the sample with the highest amplitude. High-amplitude detected samples points were matched with next ones located closer than 15 places apart, i.e. within 0.34 ms from one another, which is approximately the duration of our recorded and filtered clicks. Third, as cetaceans never abruptly change their signal amplitude within a train, it provides a consistency criterion of the click amplitude. Then, the peaks were assimilated to clicks only if their amplitude was in a range of 30 % of the weighted mean amplitude of the three preceding clicks (5/11 for the last click, 3/11 for the first and second clicks). Fourth, after an assessed click, no other click was expected during a refractory phase; the corresponding delay (1.25 to 1.7 ms), was chosen based on observed ICIs, to be long enough to prevent repeated detection of the same click, and short enough to prevent ignoring the next one.

After detecting the clicks, the program yielded the list of ICIs in milliseconds. Defects as ambient noise, omissions (a click is present but is not detected) and false alarms (program detects a click whereas there is none) were both characterized and corrected when possible or the sequence was discarded. Sequences were finally converted as chronograms, to visualize the evolution of ICI (Y-axis) in passing time (X-axis) for each click train. Despite the filtering process, some visible defects as sudden variations of the ICI for isolated points remained present in a few sequences used for analysis. These artifacts could come from the animal sound production or a false detection. Nevertheless, they were tolerated as having no incidence on the global trend of the curves. The moment of contact with the device was additionally plotted onto the graph.

Chronograms with a logarithmic Yscale provided a clear visual image of the temporal course of clicking on raw ICI data, as it emphasizes relative differences on large scales (e.g. between approach and terminal phase ICIs) as well as on small scales (within the buzz). Terminal buzzes were present. However, an unexpected profile of ICI lengthening became visible towards the end of the buzzes. Data were therefore standardized using the stop of the train, close to the moment of contact, as zero of the time axis (abscissa), a procedure similar to pre-triggering technique on an oscilloscope. ICI train profiles were then compared qualitatively on chronograms and quantitatively using parametric and non-parametric statistics. A better comparison of data issued from 36 selected sequences was prepared by standardizing on a countdown in ICI serial order.

A global image was obtained by breaking down trains into segments. For each individual train, ICIs were pooled together by successive steps of 32, beginning at the last ICI in the train and going backwards to the total number of ICIs of the sequence. The number of 32 ICIs was arbitrarily chosen as exceeding the sample size where most statistics may be interpolated [START_REF] Sokal | Introduction to Biostatistics[END_REF]. After the breakdown, a median duration was computed over the 32 ICIs of each train segment. The overall image of the 36 approach sequences was portrayed by computing, for the eight last successive steps, the minimum, first quartile, median, third quartile, and maximum of the segment's median ICI. Note that the number of segment medians (sample size) used for this image decreases from the very last, to the second -, third -, ...-, eighth last one preceding the stop, because the number of segments varies for each click train according to the total number N of ICIs in the corresponding sequence. Thus, some sequences had only one complete segment (32<N<64, the last one alone, present in all 36 sequences), whole the longest sequences had virtually many more segments one. However, medians were not computed from the initial ICI of the longest sequence, i.e. for all sequences segments. A maximum of eight segments (N>256, present in 18 sequences) were taken into consideration; beyond this maximum, data were no more present for most trains, because they were too short. Medians and other nonparametric rank statistics were privileged in order to discard other artifacts and outliers' effect.

When a phenomenon was observed abundantly in a given situation, its occurrence was tested with a binomial test to be present against a 50%-50% distribution, to attest that it occurs in most cases (more than half). When it was to be generally observed in a situation, the binomial test was conducted against a 75%-25% distribution to attest that it occurred in a vast majority of cases.

Results

Dolphins

spontaneously and profusely used echolocation towards immersed objects pointed by a familiar trainer. Above 100 min of recording provided an extensive number of echolocation sequences, with hundreds of click trains, also observed during preliminary tests.

Behavioral observations

When sent over by their trainer and after the device was immersed, the dolphins clicked and sometime glanced at their trainer from under the surface or with the head out of the water. They displayed static positioning for a brief moment or for up to ten seconds. The contact with the device occurred at the first or second presentation, generally after an observation phase staying afar. During a long static positioning on the device, dolphins sometimes repositioned themselves. The physical contact always concerned a limited area of the lower part of rostrum, no contact with any other body part was observed during the whole experiment. Except for Guama, who sometimes lifted the hydrophone for a few centimeters with its rostrum upper part (Fig. 3).

Echolocation during approach

Use of echolocation during approach. The 74 approach sequences identified on video were subdivided into 56 normal approaches (i.e. no repositioning) and 17 repositioning. Out of 56 normal approaches, 50 (89.29 %) were accompanied by a click train. Conversely, clicks were present in 2 out of 17 repositioning. Their presence significantly depended on the type of approach (χ² test (1, N= 73) = 38.25, p < 0.05), suggesting that echolocation was no longer used for minor adjustment once the target was reached.

Acceleration in phase. For each of the 36 sequences workable for ICI rhythm analysis, a chronogram was plotted that represents the ICI duration with respect to its date in passing time (Fig. 4) set with the train stop as zero. A linear trendline was adjusted to each chronogram, to determine the evolution of emission rhythm. Out of 36 trendlines, 31 showed a negative slope, which corresponded to a global acceleration in emission when the animal gets close to the target in 86.11 % of the cases, i.e., a significant majority (binomial test (.5, N=36) p < 0.05). All linear trend lines did not fit well to the data (mean R² = .36; SD= .27) because during approach the click train generally included two or more accelerations, and some decelerations. The negative trendline was attributed to a stronger speed-up while getting closer to the end of the click train (see examples on Fig. 4), as observed on 34 individual chronograms on 36. These 94.4 % of the cases are a significant large majority (binomial test (0.75, N=36) p<0.05). Thus, dolphins usually accelerated their click emission while approaching object devices, and the shorter ICIs occurred close to the end of the train. However, this acceleration was not linear, and the trend was tempered by frequent decelerations before the extreme, final speed-up, but also (as will be shown later) by some slackening-off before the train ends. Terminal buzz slackens off. Despite much inter-and intra-individual variability, compacted data from a breakdown in 32-ICI segments also demonstrated the acceleration (ICI decrease) on a global image that focuses on the termination of the approach (Figure 5). The rhythm got more than twice faster, i.e. median ICI reduced to less than half, from the 8 th lastto the very lastsegment. However, shortest ICIs were not emitted during but before the last segment.

Dolphins

decelerated their emissions (increased ICIs) just before ending their click trains. The portion of shortest median ICI, about 2ms, was typically the 3 rd or 2 nd one before the stop (32nd to 96th last ICI). This pattern seemed characteristic to our dolphins' terminal buzz. This was not an artifact due to data pooling (Fig. 6). In 32 out of 36 cases, in the last quarter of a second before the stop, chronograms fell to a minimum, after what trains ended with an increase in ICIs. This pattern of the end of the click train was actually visible on 89.89 % of the cases, a significantly large majority (binomial test, (0.75, N=36) p < 0.05). The minimum ICI value was therefore not observed at the last clik train, it generally occurred 0.25 to 0.05 second before the stop (Q3, median, Q1 = 240, 130, 54 ms respectively). When swimming closer to an object, the dolphin typically accelerated its click train; however it decelerated and stopped at the last moment of reaching the target.

Touch takes over from echolocation

Click-train ends before contact. This deceleration and the following stop were not triggered by the contact, as they occurred before it. Out of 52 times when dolphins had clicked in approach phase, 39 stopped their click train before the contact instant, and only 6 afterwards; the contact-to-stop interval remained unknown for the remaining 7. Therefore, for the 45 sure cases, 87 % had stopped their click before contact, which is a significantly large majority (binomial test (.5, N=45) p < .05). For those cases, the click train stopped for maximum 1030 ms before the contact (N = 39; M = 233 ms; SD = 331.6). If they did not stop before touching, the click train only continued for no more than 185 ms after the contact (N= 6; M = 87 ms; SD = 67.4). Overall, trains generally terminated a tenth to a hundredth of a second before the contact (Q1-, median-, Q3-delay = 9, 46, 137 ms respectively).

To sum up, dolphins usually stopped clicking just before touching the target with their rostrum. The interval between the last click and the contact was always (except for 1 sequence out of 39) greater than the last ICI before. It is not the occurrence of contact at one moment after a click in a train that makes it instantly stop. Dolphins anticipated the moment of contact.

No click while contact lasts. The stop observed on the 52 trains click trains of approach was usually definitive whether it occurred before, or exceptionally, shortly, after contact onset; for six of them only did the dolphins resume clicking before the end of positioning; in 42 cases the dolphins remained silent during the whole time contact was maintained; the 4 other cases are undetermined. Dolphins did not use echolocation simultaneously with the touch modality (binomial test (0.75, N=48) p < 0.05).

General pattern illustrated on one example

To summarize, the animal accelerated more its echolocation emission than it decelerated while approaching a non-alimentary inert object (Figure 7). As the dolphin got closer to the target, it accelerated to a maximum (minimum ICI), then slackened off and definitely stopped just before contact. The animal did not resume clicking while the contact lasted.

Discussion

Dolphins emit terminal buzz at target approach

Terminal buzz pattern of clicking was prevalent when swimming towards an object. The time course of click emission during the approach phase to a target and during the terminal phase before contact is comparable to those described for other taxa (chiropters and other odontocetes) [START_REF] Griffin | The echolocation of flying insects by bats[END_REF][START_REF] Verfuss | Echolocation by two foraging harbour porpoises (Phocoena phocoena)[END_REF][START_REF] Miller | Sperm whale behaviour indicates the use of echolocation click buzzes "creaks" in prey capture[END_REF][START_REF] Madsen | Biosonar performance of foraging beaked whales (Mesoplodon densirostris)[END_REF]. When heading to an inert target, our dolphins displayed acceleration similarly to bats, harbor porpoises and sperm-and beaked whales capturing a prey. However, the preceding searching phase, corresponding to target -or prey -research at longer distance, seemed difficult to investigate under human care conditions. This phase may be considered, with additional means, in future data sampling designs, to complete the classical threephase picture.

Functional explanations for the observed pattern are similar to those applied to other species. Global acceleration in emission increases the sampling rate, therefore the amount of collected data in a portion of time. Data may concern an object position (hence the term "echolocation") but also its shape, movement and even quality [START_REF] Harley | Bottlenose dolphins perceive object features through echolocation[END_REF]. In the wild, a rapid update of a small prey's trajectory compensates for the lack of maneuverability before attempting a catch [START_REF] Miller | Sperm whale behaviour indicates the use of echolocation click buzzes "creaks" in prey capture[END_REF]. In non-feeding situation too, the animal needs accuracy to position the rostrum on a reduced area, such as the tip of the hydrophone in our conditions. Maximal precision is required to avoid a violent contact of the skin with a target or with any obstacle, because the rostrum is so sensitive [START_REF] Mauck | Selective heating of vibrissal follicles in seals (Phoca vitulina) and dolphins (Sotalia fluviatilis guianensis)[END_REF].

In prospective phases, a neurophysiological explanation of echo processing click by click makes sense, because the ICI is much longer than the Two-Way travel Time (TWT) therefore it leaves additional time for higher brain processing. By contrast, in buzz phases, the ICI of a few seconds nears the estimated TWT. This very short interval is no longer related to the distance to the target, and it does not give enough time for more cognitive processing to interpret echoes [START_REF] Wisniewska | Buzzing during biosonar-based interceptiobn of prey in the delphinids Tursiops truncatus and Pseudorca crassidens[END_REF]. This suggests that such processing is operated on the click train as a whole, and online, i.e., during the train. It allows the dolphin to perceive and process from the acoustic radiation, the target could be mentally highlighted, allowing to obtain details not only on its position even at a very short distance (the dolphin have a visual contact of the target), but other specific information like shape, interior and exterior textures, fine movements, and if the target is alive or an object.

In short, various animals orienting towards an object or tracking a prey by echolocation pulses require speeding up the emission rate thus the terminal buzz fulfills a function of rapid and accurate update. In our four dolphins, locating and moving towards was explicitly induced by the trainers' pointing gestures to man-made targets, a very common interaction with humans for dolphins under human care. This practice most likely emerged because dolphins are prone to understand fingerand eyepointing (Pack andHerman, 2004, 2007). The observation that individuals usually kept clicking when positioning on an object already encountered, but not when repositioning on the same one after a brief loss of contact, suggests that echolocation is not limited to special novel situations, but that echolocation parameters may be stored in short-term memory.

Dolphins' terminal buzz slackens off

Our study discloses a special profile in Tursiop truncatus' terminal buzz, i.e. the very final deceleration at the end of the click train, which had not been mentioned before, in the pattern common to many different species. Anatomical studies on dolphin click generator does not suggest any explanation about emission of such ICI variations, especially at the end of the click trains [START_REF] Cranford | Functional morphology and homology in the odontocete nasal complex: implications for sound generation[END_REF][START_REF] Herzing | Social and non social uses of echolocation in free-ranging Stenella frontalis and Tursiops truncatus[END_REF][START_REF] Madsen | Single source sound production and dynamic beam formation in echolocating harbour porpoises (Phocoena phocoena)[END_REF][START_REF] Cranford | Observation and analysis of sonar signal generation in the bottlenose dolphin (Tursiop truncatus): Evidence for two sonar sources[END_REF]. Different explanations of this slackening off will be explored further studies:

Energy expenditure -Usually terminal buzzes acceleration is accompanied by a loss in amplitude [START_REF] Atem | Changes in click source levels with distance to targets: studies of free-ranging white-beaked dolphins Lagenorhynchus albirostris and captive harbour porpoise Phocoena phocoena[END_REF]. Most likely, a speed/amplitude trade-off compensates for the important cost in energy involved in higher repetition rate. In the terminal buzzes we recorded, dolphins were not only emitting quickly, but they kept emitting strongly. We think that the invested energy is important, and the mechanism of repetition cannot be stopped too abruptly [START_REF] Ridgway | Vocal reporting of echolocation targets: Dolphins often report before click trains end[END_REF]. This final slackening off is thus observed in rate, not in amplitude. However, the slackening off has not been systematically observed for each recorded approach which is weakening this assessment of mechanical constraint.

Correspondence between motion behaviour and the echolocative clicks rate -Many articles associate toothed whale buzzes with changes in the body acceleration rate [START_REF] Johnson | Beaked whales echolocate on prey[END_REF][START_REF] Miller | Sperm whale behaviour indicates the use of echolocation click buzzes "creaks" in prey capture[END_REF][START_REF] Alonso | Discrimination of fast click series produced by tagged Risso's dolphins (Grampus griseus) for echolocation or communication[END_REF]. For sperm whale, ICI variation and the whale pitch angle are correlated [START_REF] Zimmer | Combining data from a multisensor tag and passive sonar to determine the diving behavior of a sperm whale (Physeter macrocephalus)[END_REF][START_REF] Laplanche | Male sperm whale acoustic behavior observed from multipaths at a single hydrophone[END_REF][START_REF] Fais | Sperm whale predator-prey interactions involve chasing and buzzing, but no acoustic stunning[END_REF] and ICI control to target range has been observed on bottlenose dolphin [START_REF] Au | The Sonar of dolphins[END_REF]. As this sound production is used to "scan" a target or the environment, it is consistent to admit that the animal intentionally or not, modulates the sample rate (ICI) when maneuvering and interacting. The slackening off could be the consequence of a "heavy braking" just before touching the object with the rostrum.

"Victory squeal" -Ridgway et al. ( 2014) described these specific clicks before eating. He assumes that these clicks were emitted only in front of an eatable target. The acoustic features of these specific clicks are different than regular click train [START_REF] Ridgway | On doing two things at once: dolphin brain and nose coordinate sonar clicks, buzzes and emotional squeals with social sounds during fish capture[END_REF]. However, it was emitted with the strict objective to eat (or to be associated with a eatable target that it is not in our experiment). To conclude about the dolphins' echolocation slackening off: 1) it could be non-intentional due to the vocal generator itself, for anatomical or mechanical reasons the generator reduces its emission. However, if it was a mechanical relaxation phenomenon, the ICI would decrease not increase as it was the case here; 2) or it could be intentional: the dolphins adapt their ICI to the physical and spatial parameter of the target.

Click and touch

A focus on the instant when contact takes place, right after the end of the buzz, also highlights a cross-modal relationship of touch with echolocation, for perception of the same object. Prior studies have already shed some light on cross-modal integration of sonar with vision (Pack and Herman, 1995). Physical touch surely deserves attention as it is an important part of dolphins' affiliative behaviour, also sought after by humans attracted by them.

Recordings show that contact takes place with the rostrum tip, usually its lower part. This distinct posture may well favor association with sight, as eyes are positioned laterally and oriented ventrally in cetaceans. But above all, the lower jaw is the locus of reception for click echoes [START_REF] Brill | Behavioral evidence for hearing through the lower jaw by an echolocating dolphin (Tursiops truncatus)[END_REF]. This reception may be hampered by the object's direct contact thus rendering echolocation useless or not possible. Echolocation, as vision, is useful for extracting information at distance; this "distant touch" may be no more appropriate at point blank range. Either the beam originated from the melon does not reach the echoing spot, or powerful sounds reflected from too close may cause aversive reverberation to the nearlytouching rostrum. Click vibrations provoke mechanical effects: powerful clicks can be used to caress conspecifics or even to knock preys [START_REF] Herzing | Vocalizations and associated underwater behavior of freeranging Atlantice spotted dolphins, Stenella frontalis and bottlenose dolphins, Tursiops truncatus[END_REF][START_REF] Herzing | Social and non social uses of echolocation in free-ranging Stenella frontalis and Tursiops truncatus[END_REF]. Thus reciprocally, the reverberation onto the object would disturb or jam the reception of tactile stimuli. A stop in clicking that anticipates the contact guarantees the integrity of tactile stimuli and respects the sensitivity for echolocation stimuli.

Influence of the trainers' pointing gesture

The person who pointed to the target was familiar and well-known by the tested dolphins. Dolphins are used to respond to and to follow their trainers' gestures. We also know that dolphins are able of joint attention [START_REF] Pack | Bottlenose Dolphins (Tursiops truncatus) comprehend the referent of both static and dynamic human gazing and pointing in an object-choice task[END_REF]2007) so the trainer's hand gesture is informative for the animals that might comprehend it as an informative signal to go to a specific location, to look at or to look for something, to pay attention to something or to increase their vigilance towards something. To avoid this bias, the target should have been immerged in the absence of a familiar person around and then we would have recorded the spontaneous dolphins' behaviors. The presence of a familiar person giving consigns might have had some influences on the behavioral response of the dolphins. However, the trainers did not teach and/or reward the dolphins for producing clicks. Moreover, the objective of our experiment was not to interpret the clicks as dolphins do, but to analyze acoustic features, especially the variations of the ICI and intensities in order to highlight how dolphins choose to emit these click trains. Then possible biases introduced by the trainers' pointing gesture to the tested dolphin should not have impacted our results.

Conclusion

This study analyzed dolphins' behaviors and acoustical signals production while approaching an immersed non-alimentary target. According to the scientific literature on dolphins' echolocation, the present results showed that during the approach phase echolocation is followed by an acceleration of the click rate. However, unlike what has been previously demonstrated, we show here a consistent amount of cases highlighted a deceleration of the click rate, a terminal buzz slackens off, just before touching the object and no click was emitted during and after the physical contact with the object. Moreover, it seems that by processing both visual and acoustical information the dolphin gains more knowledge on the target and, particularly on the possibility to touch it without any harm, which could finally lead the dolphin to get in physical contact through its rostrum with the target. This sequential senses processing (vision and acoustics then touch) might function as a reassurance for the animal leading him/her to touch the scanned object. This strategy contributes to better understand echolocation. Dolphins produce echolocation clicks in very close proximity to the target, even if they get information from visual observation. Echolocation might not only be a distant tool that could inform about a target the dolphin cannot see, but it seems that information given by echoes reflected by the target are complementary to visual information. Echolocation gives dolphins information on the distance of the target, its size, form, outside and inside texture, density and its displacement. All this information builds a complex multi-modal image of the target. Physical stimulations engender visual, tactile, auditory sensations then after cognitive processing with emotional contents they become perceptions. Thus, one can imagine that a visual and/or acoustic stimulation (the dolphins see the target) that has generated certain sensations and perceptions is relayed (or completed) by tactile stimulation that generates a tactile sensation, itself generating a perception of the target. The emotion content is important since it has been showed that dolphin could produce a victory squeal after capturing a prey [START_REF] Dibble | Dolphins signal success by producing a victory squeal[END_REF].

Bottlenose dolphins' global accelerations and final buzz when approaching a static non-alimentary target, resemble strategies generally used by echolocating or electro-locating species performing precise actions for navigation or prey capture. In our original results, this distant touch used at close range finally slackens off until it stops shortly before the onset of full contact. Our basic gathering of click records encourages for further investigation on spontaneous echolocation behaviour in captive and wild dolphins. The sequence of pulse intervals can easily be analyzed finely, based on tools classically developed for other biological models of pulse rhythm [START_REF] Bauer | Electric organ discharge activity of resting and stimulated Gnathonemus petersii (Mormyridae)[END_REF][START_REF] Avril | Active electrolocation of polarized objects by pulse-discharging electric fish, Gnathonemus petersii[END_REF]. Clicking activity provides objective measures of bottlenose dolphins' umwelt. Moore, P., & Pawloski, D., 1990. Investigations of the control of echolocation pulses in the dolphin (Tursiops truncatus). In J. Thomas & R. Kastelein (Eds), Sensory abilities of cetaceans, New York: Plenum Press, pp. 305-320. Mullins, J., Whitehead, H., & Weilgart, L. S., 1988. Behaviour and vocalizations of two single sperm whales, Physeter macrocephalus, off Nova Scotia. Can. J. Fish. Aquat. Sci., 45(10), 1736-1743. Nowacek, D.P., 1999 Pack, A. A., & Herman, L. M., 1995. Sensory integration in the bottlenosed dolphin: Immediate recognition of complex shapes across the senses of echolocation and vision. J. Acoust. Soc. Am, 98(1), 722-733.

Fig. 1 .

 1 Fig. 1. Presented objects. The central vertical bar scales 30 cm. A, object #1: PVC disk, 2 cm-thick, 650 g. B, object #2: wooden disk; 2.5 cm-thick, 600 g. C, object #3: flexible plastic fin, 350 g. D, object #4: colored-water plastic bottle, 500 g.

Figure 2 .

 2 Figure 2. Experimental set up: (1) Trainer A on the remote platform sends one dolphin by a hand gesture to the opposite side of the pool, where it is received by trainer B on the experimental platform. (2) Experimenter C immerses the object-hydrophone device for about 30 s while B points to it. (3) The dolphin is sent back to the remote platform. Green ellipses symbolize the focus dolphin and blue squares the others.

Fig 3 .

 3 Fig 3. Examples of positioning on the object/hydrophone device. Panel A, D and E are for Guama, panels B and C for Balasi and Beauty respectively. Contacts involve the lower rostrum, except for panel D where Guama lifted the hydrophone.

Fig. 4 .

 4 Fig. 4. Examples of chronogram for each individual dolphin: ICI duration in milliseconds (log scale) as a function of countdown time from train stop in milliseconds. Linear trend lines show global acceleration for Panel A (Guama), B (Baily), C (Beauty), and D (Balasi), but not for Panel E (Balasi) because of the final deceleration. Note that linear trend lines appear as curved because of the logarithmic Y-scale. Red square is the contact onset with the object; its height shows the delay with the preceding click (Y-axis).

Fig. 5 .

 5 Fig. 5. General profile of bottlenose dolphins' clicking behaviour when arriving close to the objects. From top to bottom: maximum, third quartile, median, first quartile and minimum among N = 36 sequences, of the median ICI duration of portions grouping 32 sample ICIs. The order number of segments -and of cutpoint ICIs -is counted down with respect to the stop of the trains.

Fig. 6 .

 6 Fig. 6. Examples of terminal buzz pattern with respect to contact onset, for Guama (A and B) and Balasi (C). X-axis, countdown time from train stop, in milliseconds; Y-axis, ICI duration, in milliseconds (log scale).Red bars mark the beginning of contact with the object/hydrophone device, with red c indicating contact onset, either precisely (Panel B and C) or within a range including it (panel A).

Fig. 7 .

 7 Fig. 7. Example of click train showing the major temporal pattern emitted in approach. x-axis is countdown time from the end of the click train in milliseconds, and y-axis is the ICI duration in milliseconds (log scale).

Acknowledgements

We thank the whole dolphinarium team in Parc Astérix who gave so much time and contributed in many aspects, chose and designed the objects, and especially the curator Birgitta Mercera and the trainers Christel Villot and Deborah Mallet who participated in the experiment sessions. The program script for click detection was developped by Florent Pallas, Institut de Microélectronique Electromagnétisme et Photonique -LAboratoire d'Hyperfréquences et de Caractérisation (IMEP-LAHC, Grenoble).

Funding

This work was supported by Lyonnaise des Eaux "Partenariat environnemental et culturel » grant, ref: « Mémoire Master 2, 15 février 2012 » to E. A.

List of abbreviations

ICI = Inter click interval TWT = Two-Way Time


Florent Olivon

Pascal Retailleau

Sandy Desrat

David Touboul

Fanny Roussi

Cécile Apel

Marc Litaudon

Isolation of Picrotoxanes from Austrobuxus carunculatus Using Taxonomy-based Molecular Networking

ABSTRACT: A unique collection of 292 extracts from 107 New Caledonian Euphorbiaceae species sensu lato was profiled by LC-MS 2 and the metabolite content organized by molecular networking. Based on the assumption that taxon-specific molecules are more likely to be structurally novel, taxonomic data were mapped on spectral networks to detect genusspecific clusters. Using this approach, a group of compounds unique to the genus Austrobuxus was highlighted. The subsequent MS-guided purification of the fruit EtOAc extract of Austrobuxus carunculatus led to the isolation of 13 new monolactone and "norditerpene" picrotoxanes (2-14), along with the known tutin (1). The structures of the new compounds were elucidated by HRESIMS and NMR spectroscopic data analysis, and the absolute configurations of compounds 1, 3, 7, 11, 12 and 14 were determined by singlecrystal X-ray diffraction analysis. The relative and absolute configurations of compounds 4 and 5 were ascertained by chemical transformation of compound 3. The absolute configurations of other members of the series have been proposed on the basis of biogenetic considerations and specific rotation values of similar sign and magnitude.

Compounds 1-14 were evaluated for their antiproliferative activities against HCT116 colon, U87-MG glioblastoma and A549 lung human cancer cell lines. Compounds bearing an acyl chain at C-2 (i.e. 2, 4 and 13) showed IC50 values in the micromolar range for the three cell lines used.

It is now widely accepted that the occurrence of specialized metabolites (SM) within the plant kingdom and their exceptional chemical diversity is the result of evolutionary processes. [1][2][3] Millions of years of environmental screening have endowed natural products with structures and functions shaped and optimized to overcome abiotic stress and fight natural enemies. Chemosystematics aims at studying the distribution of metabolites within the plant kingdom and has always relied on botanical classification. However, it has been shown that within the same botanical family, certain taxa biosynthesize SM that are or become specific to their producer. Based on the postulate that different groups of SM may have an identical ecological role, their occurrence within a clade is sometimes mutually exclusive, in order to maintain an advantageous cost/benefit ratio. 4 As a result, a single and structurally homogeneous group of SM may dominate within a given taxon. This unequal distribution between tribes or genera has even been used to build the classification of certain plant families. 5 Based on this observation, a methodology identifying genus-or species-specific metabolites has been developed, with the ultimate goal of discovering novel structures or scaffolds. The present prioritization strategy consists of adding taxonomic data to a spectral network resulting from a tandem mass spectrometry data organizational approach. [6][7][8][9] The rapid detection of taxon-specific clusters and subsequent dereplication have helped guide further work on the most promising extracts.

The work described herein is in continuation of the chemical exploration of an extract collection of 292 New Caledonian Euphorbiaceae species sensu lato initiated in the abovementioned studies. [6][7][8] The currently accepted classification of Euphorbiaceae sensu lato by the Angiosperm Phylogeny group recognizes five different families: Euphorbiaceae Juss. sensu stricto, Pandaceae Engl. & Gilg, Phyllanthaceae Martynov, Picrodendraceae Small and Putranjivaceae Endl.. 10 From this collection, the approach used enabled the detection of ion clusters specific to the genus Austrobuxus Miq.. The corresponding compounds were particularly abundant in the fruit extract of the species Austrobuxus carunculatus (Baill.) Airy Shaw. A. carunculatus, for which no chemical investigation has been reported so far, belongs to the family Picrodendraceae and is endemic to New Caledonia. The MS-guided purification of its fruit EtOAc extract led to the isolation of 13 new picrotoxanes: 1 monolactone (2) and 12 structurally related "norditerpenes" (3-14), along with the known tutin (1).

Picrotoxanes are found mainly as tetra-or pentacyclic complex structures containing up to 12 stereogenic centers. [START_REF] Gössinger | In Fortschritte der Chemie organischer Naturstoffe / Progress in the Chemistry of Organic Natural Products[END_REF] Most members of this chemical class are neurotoxins acting as GABA-receptor antagonists, [START_REF] Hosie | [END_REF][13][14][15][16][17][18][19] and are used as tools in neurobiological research. So far, the "norditerpene" structural variant of the picrotoxanes is restricted to the family Picrodendraceae. [START_REF] Gössinger | In Fortschritte der Chemie organischer Naturstoffe / Progress in the Chemistry of Organic Natural Products[END_REF] With reference to compounds isolated from Austrobuxus swanii, 20 the presently isolated compounds were named austrobuxusins E-M (2, 4-6, 9, 10, 12-14) and 16epi-austrobuxusins B, G and H (11, 8 and 7, respectively). Compound 3 was named 16-epipretoxin. The present report describes their isolation, structure elucidation and cytotoxic activity evaluation using the HCT116, A549 and U87-MG cell lines.

RESULTS AND DISCUSSION

A collection of 292 EtOAc extracts was prepared and profiled by UHPLC-HRMS 2 as previously described. [6][7][8] The set comprised EtOAc extracts of different plant parts prepared from 107 New Caledonian species (93% are endemic) coming from 20 genera, of which 40% were Euphorbiaceae sensu stricto, 46% Phyllanthaceae, 13% Picrodendraceae and 1% Putranjivaceae. The Pandaceae family is not represented in New Caledonia and is therefore not part of the sample series used. Spectral data were processed via MZmine 2 21 before being organized as molecular networks using MetGem. 22 A multi-informative molecular map was created by overlaying color-coded taxonomic data on the initial networks. For this purpose, the extracts were grouped according to the genus they belong. The color mapping consisted of assigning a specific color to each group, so as to easily differentiate multicolored ion clusters (for which the corresponding ubiquitous compounds come from multiple taxa), from single-colored clusters, which are therefore genus or species-specific. Using this approach, several clusters bringing together protonated ions specific to the genus Austrobuxus were highlighted (Figure 1). These clusters were separated and scattered in the classical representation of molecular networks (Figure 1A), while they were well gathered on the t-SNE visualization along with other isolated nodes (Figure 1B). The latter preserves all relations between groups of spectra and allows the gathering of sub-structurally related compounds.

O O R 1 O O OH O O R 2 O H
3 R 1 = H, R 2 = H, α H-16 4 R 1 = CO(C 11 H 23 ), R 2 = H, α H-16 5 R 1 = H, R 2 = CO(C 11 H 23 ), α H-16 6 R 1 = H, R 2 = CO(C 9 H 19 ), α H-16 7 R 1 = H, R 2 = CO(C 9 H 19 ), β H-16 8 R 1 = H, R 2 = CO(C 11 H 23 ), β H-16 9 R 1 = H, R 2 = CO(C 13 H 27 ), β H-16 10 R 1 = H, R 2 = CO(C 15 H 31 ), β H-16 R 2 O O R 1 O O OH O O HO 11 R 1 = H, R 2 = isopropenyl 12 R 1 = H, R 2 = isopropyl 13 R 1 = CO(C 9 H 19 ), R 2 = isopropyl O O RO O O OH 1 R = H (tutin) 2 R = CO(C
When analyzing a large sample set, t-SNE parameters can be modulated for easier browsing within the graph (i.e. to avoid overloading and obtain a clearer delimitation of the different clusters). Nodes can be filtered to keep only those that interact with a representative number of other nodes: in the representation Figure 1B, nodes were retained when sharing at least 6 cosine scores above 0.5. Spectral similarity information is maintained since the filter applies to nodes, not cosine scores.

None of the compounds within the specific clusters could be annotated using the GNPS libraries. 23 However, database searching from predicted molecular formulas, together with taxonomic considerations, suggested the presence of pretoxin or its isomers. 24 Despite a moderate ranking (22 nd candidate considering the CSI:FingerID bio database), this information was corroborated by using Sirius. 25,26 The new compounds of interest were detected as present mainly in the fruit EtOAc extract of Austrobuxus carunculatus. Its MS-guided chemical investigation led to the isolation of 14 picrotoxanes for which the structures were elucidated by HRESIMS and NMR spectroscopic data analysis (Tables 1234, Table S1 and S3, Supporting Information). Their absolute configurations were determined by single-crystal X-ray diffraction (S2, Supporting Information), semi-synthesis and on the basis of biogenetic considerations. Compounds 1, 2, 13 and 14 were not detected within the clusters of interest but co-purified during the targeted isolation of compounds 3-12. Compound 1 was identified as the known tutin 27 picrotoxane on the basis of the data obtained. Its HRESIMS data showed a [M-H2O+H] + ion at m/z 277.1107 (calcd for C15H17O5 + , 277.1081) corresponding to the molecular formula C15H18O6. The combination of 1D-and 2D-NMR data, as well as X-ray diffraction providing some evidence regarding the absolute configuration, allowed the confirmation of its characteristic five-fused rings and their substituents (See Table S1, Supporting Information for NMR data and Figures 2 and S2.2

Supporting Information for X-ray ORTEP view [START_REF] Burnett | ORTEP-III: Oak Ridge Thermal Ellipsoid Plot Program for Crystal Structure Illustrations[END_REF][START_REF] Macrae | [END_REF] and overlay diagram for the original KEGBAZ 30 and the present model of 1). Analysis of its data allowed compound 3 to be identified as an epimer of the known pretoxin. 24 In contrast to monolactone picrotoxanes such as compounds 1 and 2, the so-called "norditerpene" picrotoxanes possess an extended carbon skeleton. The main difference between compounds 1 and 3 was the presence of a substituted spiro g-butyrolactone at C-13 in 3 instead of the spiro-epoxy moiety in 1. The structure and absolute configuration of 3, which was recrystallized from MeOH, was confirmed unambiguously by X-ray diffraction data analysis. (See Table 1 for the NMR data and Figures 2 and S2 The structure of compound 6 only differed from compound 5 by the possession of a shorter acyl chain at C-18, comprising 10 instead of 12 carbon atoms. Its absolute configuration may be assumed to be the same as that of compounds 3-5 on the basis of biogenetic considerations and their specific rotation values of similar sign and magnitude.

The analysis of NMR spectroscopic data of compound 7 indicated its structure as being very close to those of 5 and 6. However, slight differences in 1 H NMR resonances of the methines H-12, H-16 and H-18, the methylene protons H2-14, and the methyl protons H3-7

suggested a different stereochemistry. Indeed, the relative configuration of C-16 was established on the basis of strong ROESY correlations between H-16, H-12 and H-14-b, all oriented above the mean plane. Thus, compound 7 was assigned as the epimer at C-16 of compound 6. Crystals suitable for single X-ray analysis, obtained by vapor diffusion from methanol against hexane, were used to confirm its structure and absolute configuration (See The HRESIMS data of compound 13 showed a [M+H] + ion peak at m/z 567.3195 (calcd for C30H47O10 + , 567.3175) corresponding to the molecular formula, C30H46O10. The NMR spectroscopic data of 13 generally were comparable to those of compound 12, but revealed the presence of a C10-saturated-carbon ester side chain at C-2 instead of the hydroxy group present in 12. The same key COSY and HMBC correlations that supported the assignments of the ester chains at C-2 in 2 and 4 were also observed in 13 and were used to establish the structure as depicted. Crystallographic analysis confirmed the relative configuration of the molecule but the single crystals obtained were of insufficient quality to determine its absolute configuration (see S2.2, Supporting Information for the ORTEP view of 13). However, this is assumed to be identical as that of compounds 11 and 12 based on their common biogenesis.

The HRESIMS data of compound 14 showed a protonated molecular ion at m/z 363.1467

[M+H] + (calcd 363.1449), corresponding to the molecular formula, C19H22O7. The molecular structure consisted of the "norditerpene" picrotoxane skeleton comprising an ethylidene gbutyrolactone. The compound 14 could be formed from dehydration of 16-epi-pretoxin (3).

Compounds 1-14 were evaluated at 1 and 10 µM for their antiproliferative activities against the HCT116 colon, U87-MG glioblastoma, and A549 lung human cancer cell lines. No compound showed cytotoxicity at 1 µM. However, compounds 2, 4 and 13 exhibited discernible cytotoxic activities at 10 µM for all three cell lines. Their subsequent evaluations at 10 different concentrations allowed IC50 values to be determined and were found to be in the micromolar range. In particular, compounds 4 and 13 showed IC50 values of 5.8 ± 0.5 µM for A549 cell line and 0.7 ± 0.1 µM for U87-MG cell line, respectively. Interestingly, compounds 2, 4 and 13 are the only ones in the series that share a common acyl chain at C-2, which appears to be essential for cytotoxic activity for the cell lines evaluated.

The application of the presently used methodology in combining spectral and taxonomic data facilitated the isolation of 13 new picrotoxane-type compounds. The MetGem dual display offers two complementary visualizations, and is even more useful when studying large sets of samples. While the standard molecular networks representation may appear clearer and specific clusters can be easily located/defined, the t-SNE organization provides additional information by joining clusters and isolated nodes related to a larger group. The detection of taxon-specific clusters should be associated with a comprehensive dereplication step because specificity does not necessarily imply compound novelty or originality. In this regard, it is helpful to share MS 2 information in order to enrich databases with new experimental spectra. MS 2 data from most of the compounds isolated in this work were deposited in the GNPS public library accordingly (GNPS IDs can be found in the Experimental Section). These data are valuable for direct annotation of features via library searching, to train and refine computational metabolite annotation algorithms and to optimize the efficiency of annotation workflows that also consider orthogonal information (molecular network topology, predicted retention times, taxonomy, etc.). [32][33][34] This is all the more important for structural classes not represented in spectral databases, as seems to be the case for picrotoxanes.

Furthermore, the efficiency and relevance of the methodology used greatly depend on the constitution of the sample series studied. When studying a botanical family or a phylogenetically related group, as was the case in the present study, it is beneficial to bring together as many different genera (or subsets) as possible. By covering the largest molecular extent within these groups, this allows for more reliable observations of their ubiquitous chemical spaces and thus, avoids the emergence of "falsely taxon-specific" clusters. The present collection contains a non-homogeneous distribution of Picrodendraceae representatives (Austrobuxus, Longetia and Scagea, i.e, the only three Picrodendraceae genera present in New Caledonia). Consequently, the picrotoxanes isolated in this work were detected as specific to the genus Austrobuxus, but their occurrence within several other Picrodendraceae genera is known. [START_REF] Gössinger | In Fortschritte der Chemie organischer Naturstoffe / Progress in the Chemistry of Organic Natural Products[END_REF] These compounds are nonetheless structurally fascinating and it would have been interesting to have greater sampling allowing a finer distinction to be made between the different types of picrotoxanes and their distribution within the Picrodendraceae.

Taxa originally belonging to the Euphorbiaceae have been reclassified into five different families. In particular, based on rbcL gene sequences analysis, Savolainen et al. elevated the Euphorbiaceae subfamily Oldfieldioideae to the Picrodendraceae (as the Pseudanthaceae). 35 The isolation of "norditerpene" picrotoxanes in Austrobuxus carunculatus appears to be consistent with this recent taxonomic revision, as this scaffold has only been reported in species in the Picrodendraceae. By visualizing chemical contents and highlighting specific molecular families, the molecular networking approach has facilitated the detection of chemotaxonomic markers, and thus appears as a convenient tool to support botanical classifications. has been deposited at the Herbier IRD de Nouméa, New Caledonia. Fruits were cut before being dried using a hot air generator (the temperature did not exceed 40 °C) and stored at room temperature.

EXPERIMENTAL SECTION

Extraction and Isolation. The plant material (1.3 kg, dry wt.) was extracted three times for 3 hours at room temperature with EtOAc (2 L each). The EtOAc extract was concentrated in vacuo at 40 °C. This extract was dissolved in 0.5 L of CH3CN and subjected to a liquid/liquid partition process with n-heptane (2 × 0.5 L) to afford 7.2 g of a CH3CN-soluble fraction. This residue was subjected to silica gel column flash chromatography (120 g) using a gradient of n-heptane-EtOAc-MeOH of increasing polarities (100:0:0 to 0:100:0 to 0:80:20, 50 mL.min -1 ) to afford 33 fractions, F1-F33, according to their TLC profiles. F12 (98 mg) was purified by silica gel column flash chromatography (12 g) using a gradient of n-heptane-EtOAc furnished compounds 11 (18.9 mg, tR 6.5 min) and 12 (21.0 mg, tR 7.9 min). F22 (736.2 mg) was purified by silica gel column flash chromatography (80 g) using a gradient of n-heptane-EtOAc (60:40 to 50:50, in 20 min) leading to fractions F22-1 and F22-2. F22-1 (468.2 mg) was separated by semi-preparative HPLC (Kinetex C18, H2O-CH3CN 75:25 at 4.5 mL.min -1 ) to yield compound 3 (207.3 mg, tR 6.4 min).

(1R,2S,3R,4R,5S,6R,11S,12R,13R)-Tutin (1): white needles;

[𝛼] $ %& +14 (c 1, MeOH); 1 H and 13 C NMR, see Table S1 36 All .mzXML were then processed using MZmine 2 v31. 21 The mass detection was conducted using a noise level of 500 counts for MS and 50 counts for MSMS dimension. The ADAP chromatogram builder was used with a minimum group size of scans of 5, a group intensity threshold of 1000, a minimum highest intensity of 2000 and a m/z tolerance of 10 ppm. 37 The ADAP wavelets deconvolution algorithm was used with the following standard settings: S/N threshold = 7, minimum feature height = 2500, coefficient/area threshold = 25, peak duration range 0.05 -0.5 min, RT wavelet range 0.01 -0.1. However, considering the diverse molecular contents of extracts included within this wide-ranging collection, these parameters needed to be re-optimized for a few specific samples showing divergent chromatographic profiles and peak shapes. MS 2 scans were paired using a m/z tolerance range of 0.01 Da and RT tolerance range of 0.1 min. Isotopologues were grouped using the isotopic peaks grouper algorithm, with a m/z tolerance of 5 ppm and a RT tolerance of 0.1 min. Peak alignment was performed using the join aligner module (m/z tolerance = 5 ppm, weight for m/z = 2, weight for RT = 1, absolute RT tolerance 0.2 min).

The peak list was gap-filled with the same RT and m/z range gap filler module (m/z tolerance of 5 ppm. Eventually, the .mgf spectral data file and its corresponding .csv metadata file (containing RT and peak areas) were exported using the dedicated "Export to GNPS" built-in modules. 38 The .mgf file was imported into Sirius 4.4.17 and spectra of interest were processed using the Molecular Formula Identification and the CSI:FingerID modules. 25,26 The MS2 MassDev parameter was set to 5 ppm and only the bio database was considered for structure prediction.

Molecular Network Analysis. The two files mentioned above were imported into MetGem. 22 MS 2 spectra were window-filtered by choosing only the top six peaks within the ± 50 Da window throughout the spectrum. The data were filtered by removing all peaks in the ± 17 Da range around the precursor m/z. The m/z tolerance windows used to find the matching peaks was set to 0.01 Da, and cosine scores were kept under consideration for spectra sharing 5 matching peaks at least. The standard molecular networks were created where edges were filtered to have a cosine score above 0.7. Further edges between two nodes were kept in the network only if each of the nodes appeared in each other's respective top 10 most similar nodes.

Regarding the t-SNE output, nodes were kept under consideration only if they shared at least 6 cosine scores above 0.5 with others ("at least 6 cosine scores above 0.5" parameter).

The number of iterations, perplexity, learning rate, and early exaggeration parameters were set to 10000, 10, 150, and 12, respectively. The Barnes-Hut approximation was activated using a θ angle of 0.2°.

Both standard molecular networks and t-SNE outputs were imported in Cytoscape 3.

ASSOCIATED CONTENT Supporting Information

1 H and 13 C NMR chemical shifts of compound 1, X-ray analyses experimental details, ORTEP views, Crystal data, Structure refinement and Conventional hydrogen bonds of Graphical Abstract:

Figure 1 .

 1 Figure 1. Multi-informative molecular maps generated with MetGem. Networks were

Figure 2 . 30 % for 14 )

 23014 Figure 2. ORTEP views of compounds 1, 3, 7, 11, 12 and 14. (Ellipsoids drawn at 50% (30

  .2, Supporting Information for X-ray ORTEP representation of 3). Compound 3 is 16-epi-pretoxin. In the literature, the absence of crystallographic data and complete NMR data for pretoxin did not allow their comparison with those of compound 3. Only the melting points of the two compounds could be compared and proved to be different, confirming their structural difference (Mp = 128-130°C24 and 208-210°C for pretoxin and 16-epi-pretoxin, respectively).The HRESIMS data of compound 4 showed a [M+H] + ion peak at m/z 563.3176 (calcd for C31H47O9 + , 563.3215), corresponding to the molecular formula, C31H46O9. The NMR spectroscopic data showed the presence of similar structural features to those of 16-epipretoxin. However, compound 4 was found to possess a C12-saturated-carbon ester side chain at C-2 instead of a hydroxy group as in 16-epi-pretoxin. The MS 2 spectrum of the sodiumcationized molecule showed a fragment ion corresponding to a neutral loss of a C12 fatty acyl group followed by the loss of a molecule of water, which confirmed the nature of the side chain. As for compound 2, bearing the same acyl chain at C-2, key COSY, HMBC and ROESY correlations were used to validate the structure as depicted. However, there was some doubt about the relative configuration of C-18 in the side chain of 4 connected to C-16.To resolve this uncertainty, the semi-synthesis of compound 4 was carried out from compound 3, for which the absolute configuration is known. Esterification of 3 by lauric acid afforded the semi-synthetic compound of interest, with the 1 H NMR spectrum along with the specific rotation value ([𝛼] $ %& +12, c 0.1, MeOH) being identical to those of compound 4, confirming their identical relative and absolute configurations (See S3, Supporting Information for comparison of the 1 H NMR spectra). Compound 5 exhibited the same molecular formula and shared very similar data to 4. The only structural difference between 5 and 4 was the attachment of the ester side-chain to C-18 instead of C-2 in 4, as suggested by the HMBC correlation between H-18 and C-1′. The presence of an acyl chain at C-18 resulted in the downfield shift of protons H-16 and H-18 resonances (dH 2.70 to 3.15 and 3.94 to 5.06, respectively). As for compound 4, the relative and absolute configurations of compound 5 were determined from the identical 1 H NMR spectroscopic data and optical rotation values between the semi-synthetic and natural compounds (See S3, Supporting Information for comparison of the 1 H NMR spectra).

Figure 2 and S2. 2 , 10 .

 2210 Figure 2 and S2.2, Supporting Information for the X-ray ORTEP view of 7).

1 - 4 , 6 , 7 and 9 - 14 .

 146914 Experimental Procedures. Optical rotations were measured at 20 °C on an Anton Paar MCP 300 polarimeter. Melting points were measured with a Büchi Melting Point B-540 apparatus. NMR spectra were recorded on a Bruker 300 MHz instrument (Avance 300) for compounds 5 and 8 and on a Bruker 500 MHz instrument (Avance 500) for compounds Chemical shifts (relative to CDCl3 or CD3OD for 1, 3 and 12) are in ppm and coupling constants are in Hz. Kinetex analytical and semi-preparative C18 columns (250 × 4.6 mm and 250 × 10 mm; 5 μm Phenomenex) were used for HPLC separations using a Waters autopurification system equipped with a sample manager (Waters 2767), a column fluidics organizer, a binary pump (Waters 2525), a UV-vis diode array detector (190-600 nm, Waters 2996), and a PL-ELS 1000 ELSD Polymer Laboratory detector. HRMS data were acquired using an Acquity Waters UPLC coupled to a Waters LCT Premier XE mass spectrometer. The UPLC system was equipped with a Waters Acquity PDA dectector. Separation was achieved on a BEH C18 column (1.7 μm, 2.1 mm × 50 mm) at a flow rate of 0.6 mL.min -1 . Elution was conducted with a H2O-CH3CN + 0.1% formic acid gradient as follows: 95:5 to 0:100 in 5.5 min. The ionization was carried out using an electrospray ionization source in the positive mode (range m/z 80-1500). All solvents were purchased from Carlo Erba (France) or SDS (Peypin, France) and analytical plates (Si gel 60 F254) were from Merck (France). Prepacked GraceResolv silica cartridges were used for flash chromatography using a Teledyne Isco Combiflash Rf 200i. Plant Material. Fruits of Austrobuxus carunculatus were collected at Port Boisé (New Caledonia) in May 1998 and authenticated by M. Litaudon. A voucher specimen (LIT-0538)

  (95:5 to 60:40, in 15 min) to afford compound 2 (71 mg). F14 (86.5 mg), F15 (760.2 mg) and F16 (231.8 mg) were subjected to semi-preparative HPLC (Kinetex C18, H2O-CH3CN 15:85 (F14), H2O-CH3CN 25:75 for 10 min to H2O-CH3CN 0:100 in 10 min (F15), H2O-CH3CN 20:80 (F16) at 4.5 mL.min -1 ) to afford compound 13 (8.5 mg, tR 6.9 min), compounds 5-10 (101.5 mg, tR 11.3 min; 15.5 mg, tR 7.3 min; 16.8 mg, tR 6.7 min; 159.0 mg, tR 9.6 min; 108.7 mg, tR 16.2 min; 26.3 mg, tR 20.8 min) and compound 4 (79.7 mg, tR 9.2 min), respectively. F18 (124.4 mg) was purified by silica gel column flash chromatography (12 g) using a gradient of n-heptane-EtOAc (80:20 to 50:50, in 20 min) leading to four fractions, F18-1 to F18-4. F18-2 (69.2 mg) was separated by semi-preparative HPLC (Kinetex C18, H2O-CH3CN 75:25 at 4.5 mL.min -1 ) to yield compounds 1 (13.0 mg, tR 5.0 min) and 14 (14.5 mg, tR 12.2 min). Purification of F20 (215.8 mg) by semi-preparative HPLC (Kinetex C18, H2O-CH3CN 75:25 at 4.5 mL.min -1 )

7 . 2

 72 for advanced network visualization and image export. 39 Data availability. Raw .d and .mzXML files of the EtOAc fruit extract of Austrobuxus carunculatus were deposited in the MassIVE data repository (MassIVE ID: MSV000085524). Raw .d files of the 292 extracts, .mgf and .csv output files, and molecular networks files are also available on MassIVE (MassIVE ID: MSV000086113).Cell Culture and Proliferation Assay. Cancer cell lines were obtained from the American Type Culture Collection (Rockville, MD, USA) and were cultured according to the supplier's instructions. Human HCT-116 colorectal carcinoma cells were grown in Gibco McCoy's medium 5A supplemented with 10% fetal calf serum (FCS) and 1% L-glutamine. Human A549 lung cancer cells were grown in RPMI 1640 supplemented with 10% FCS and 1% Lglutamine. Human U87-MG glioblastoma cells were grown in DMEM supplemented with 10% FCS and 1% L-glutamine. Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2.Cell viability was determined by a luminescent assay according to the manufacturer's instructions (Promega, Madison, WI, USA). For IC50 determinations, the cells were seeded in 96-well plates (3 × 10 3 cells/well) containing 90 μL of growth medium. After 24 h of culture, the cells were treated with the test compounds at 10 different final concentrations. Each concentration was obtained from serial dilutions in culture medium starting from the stock solution. Control cells were treated with the vehicle. Experiments were performed in triplicate.After 72 h of incubation, 100 μL of CellTiter Glo Reagent were added for 15 min before recording luminescence with a spectrophotometric plate reader PolarStar Omega (BMG LabTech). The dose-response curves were plotted with Graph Prism software and the IC50 values were calculated using the Graph Prism software from polynomial curves (four or fiveparameter logistic equations). Doxorubicin was used as a positive control with IC50 values of 56.6 ± 0.8, 99.6 ± 2.3 and 90.0 ± 3.0 nM for HCT116, U87-MG and A549 cell lines, respectively.

Table 1 . 1 H and 13 C NMR Data for Compounds 2-5 in CDCl3 (3 in CD3OD)

 1

	Compound 2 gave the molecular formula C27H40O7, as indicated by HRESIMS of the [M+H] +
	ion peak at m/z 477.2828 (calcd for C27H41O7 + , 477.2847). Its NMR spectra displayed similar
	proton and carbon resonances and cross correlations with those of tutin indicating the
	presence of the same a-isopropenyl group at C-4 on the cyclohexane ring, the ester bridge
	between C-3 and C-5 forming the g-lactone ring, the a-methyl group at C-1 as well as the a-
	hydroxy group at C-6. Compound 2 also contained a cis-fused a-epoxy function located in
	the C-11/C-12 positions and a spiro-epoxy moiety at C-13 on the cyclopentane ring. The
	major structural difference between compound 2 and tutin was the presence of a C12-saturated-
	carbon ester side chain instead of a hydroxy group at C-2. This was supported by the
	presence of an ester carbonyl resonance at d 173.8, which correlated with the H-2 methine
	proton at dH 5.33 (s, H-2) and two methylene resonances at dH 2.33 (m, H2-2′) and 1.61 (m, H2-
	3′) in the HMBC spectrum. The small deshielding effect observed on H-2 was consistent with
	the substitution of tutin C-2 hydroxy group by an ester moiety. The relative configuration of
	C-2 was deduced from the ROESY correlations observed between H-2 and H-3, H3-7 and the
	olefinic proton H-9b of the isopropenyl group, indicating the b-orientation of the long-chain
	ester moiety. The absolute configuration of 2 is assumed to be the same as that of tutin on the
	basis of biogenetic considerations and their specific rotation values of similar sign and
	magnitude.

29.1-29.9 1.17-1.34 m 29.1-29.7 1.15-1.35 m 29.1-29.9 1.15-1.30 m 8′ 29.1-29.9 1.17-1.34 m 29.1-29.7 1.15-1.35 m 29.1-29.9 1.15-1.30 m 9′ 29.1-29.9 1.17-1.34 m 29.1-29.7 1.15-1.35 m 29.1-29.9 1.15-1.

Table 2 . 1 H and 13 C NMR Data for Compounds 6-9 in CDCl3

 2

			6		7		8		9
	position	δC	δH (J in Hz)	δC	δH (J in Hz)	δC	δH (J in Hz)	δC	δH (J in Hz)
	1	54.2	-	52	-	52	-	52	-
	2	73.7	4.29 brs	74.2	4.34 brs	74.2	4.34 brd (4.4)	73.7	4.30 brs
	OH-2	-	-	-	-	-	-	-	-
	3	82.6	4.82 brd (4.7)	82.5	4.84 brd (4.9)	82.5	4.84 brd (4.8)	82.8	4.80 brd (4.1)
	4	49.8	3.19 m	49.8	3.19 m	49.8	3.19 m	49.7	3.15 m
	5	48.7	3.11 d (4.4)	48.9	3.10 d (4.2)	48.9	3.10 d (4.3)	48.9	3.07 d (3.6)
	6	77	-	76.9	-	76.9	-	76.8	-
	OH-6	-	2.48 brs	-	2.49 brs	-	2.73 brs	-	2.76 s
	7	23.9	1.23 s	23.6	1.34 s	23.6	1.34 s	23.5	1.30 s
	8	141	-	141	-	141	-	141	-
	9-a	111.9	4.93 brs	112.2	4.94 brs	112.2	4.94 brs	112	4.90 brs
	9-b		4.69 brs		4.69 brs		4.69 brs		4.68 brs
	10	23.1	1.88 s	23	1.88 s	23	1.88 s	23	1.85 s
	11	61.9	3.75 d (2.7)	61.9	3.73 d (2.8)	61.9	3.74 d (2.8)	61.8	3.70 brs
	12	64.1	3.35 d (2.7)	61.8	3.44 d (2.8)	61.8	3.44 d (2.8)	61.6	3.43 brs
	13	90.6	-	90.5	-	90.5	-	90.7	-
	14-α		3.61 dd (14.3,	30.3	3.62 dd (13.9, 11.5)	30.2	3.62 dd (13.9, 11.5)	30.4	3.61 dd (13.5, 11.6)
		31.3	11.2)
	14-β		1.82 dd (14.3,		2.32 dd (13.9, 9.6)		2.32 dd (13.9, 9.6)		2.29 dd (13.5, 9.6)
			7.9)
	15	174.2	-	174.5	-	174.5	-	174.9	-
	16	45.4	3.15 ddd (11.2,	45.9	2.87 ddd (11.5, 9.6,	45.9	2.87 ddd (11.5, 9.6,	45.8	2.87 ddd (11.6, 9.6,
			7.9, 3.9)		5.5)		5.5)		5.3)
	OH-16	-	-	-	-	-	-	-	-
	17	176.2	-	174.7	-	174.7	-	174.9	-
	18		5.06 qd (6.5,	68.8	5.20 qd (6.4, 5.5)	68.8	5.20 qd (6.4, 5.5)	69	5.13 qd (6.4, 5.3)
		70.3	3.9)
	OMe-18 -	-	-	-	-	-	-	-
	19	17.1	1.37 d (6.5)	18.4	1.36 d (6.4)	18.4	1.36 d (6.4)	18.3	1.32 d (6.4)
	1′	173.5	-	173.4	-	173.4	-	173.6	-
	2′	34.7	2.26 m	34.7	2.25 t (7.6)	34.7	2.25 t (7.6)	34.6	2.22 t (7.2)
	3′	25.1	1.58 m	25.1	1.58 m	25.1	1.58 m	25.1	1.55 m
	4′-7′	29.35 m
	10′	14.3	0.85 t (6.9)	14.3	0.85 t (6.9)	32.1	1.20-1.35 m	29.3-29.9 1.15-1.35 m
	11′					22.8	1.20-1.35 m	29.3-29.9 1.15-1.35 m
	12′					14.3	0.85 t (6.9)	32.1	1.15-1.35 m
	13′							22.8	1.15-1.35 m
	14′							14.2	0.83 t (6.9)

3-29.7 1.20-1.32 m 29.3-29.7 1.15-1.35 m 29.3-29.9 1.20-1.35 m 29.3-29.9 1.15-1.35 m 8′ 32.1 1.20-1.32 m 32.1 1.15-1.35 m 29.3-29.9 1.20-1.35 m 29.3-29.9 1.15-1.35 m 9′ 22.9 1.20-1.32 m 22.9 1.15-1.35 m 29.3-29.9 1.20-1.35 m 29.3-29.9 1.15-1.

Table 3 . 1 H and 13 C NMR Data for Compounds 10-13 in CDCl3 (12 in CD3OD)

 3

			10		11		12		13
	position	δC	δH (J in Hz)	δC	δH (J in Hz)	δC	δH (J in Hz)	δC	δH (J in Hz)
	1	52	-	52.1	-	53.3	-	51.3	-
	2	74.1	4.33 brs	74.4	4.31 brs	75.4	3.77 brs	75.4	5.00 brs
	OH-2	-	-	-	3.03 brs	-	-	-	-
	3	82.6	4.84 brd (4.9)	82.6	4.86 brd (4.9)	85.4	4.58 brs	80.5	4.61 brd (4.2)
	4	49.8	3.18 m	49.8	3.18 m	54.7	2.17 m a	53.7	2.18 m a
	5	48.9	3.10 d (4.3)	48.9	3.10 d (4.2)	50.4	2.84 brs	48	2.93 d (3.3)
	6	76.9	-	77.1	-	78.4	-	77.8	-
	OH-6	-	2.52 s	-	2.57 s	-	-	-	-
	7	23.6	1.34 s	23.6	1.37 s	24.1	1.40 s	23.1	1.34 s
	8	141	-	141	-	28.1	2.17 m a	27.3	2.18 m a
	9-a	112.1	4.94 brs	112.1	4.94 brs	22.4	1.06 d (4.0)	22.1	1.04 m a
	9-b		4.69 brs		4.68 brs
	10	23	1.88 s	23.0	1.88 s	20.6	0.92 d (4.0)	20.5	1.04 m a
	11	61.9	3.73 d (2.7)	61.8	3.72 d (2.8)	63.1	3.64 brs	62.1	3.72 d (2.8)
	12	61.7	3.44 d (2.7)	62.6	3.95 d (2.8)	62.9	3.93 brs	62.5	4.20 d (2.8)
	13	90.5	-	91.1	-	93.7	-	91.4	-
	14-a	30.3	3.62 dd (13.9,	36.3	3.65 d (15.1)	37.4	3.94 d (14.3)	36	3.39 d (14.4)
			11.5)
	14-b		2.32 dd (13.9,		2.16 d (15.1)		2.00 d (14.3)		2.18 m a
			9.5)
	15	174.7	-	174.7	-	177.8	-	174.9	-
	16	45.9	2.87 ddd (11.5,	79.1	-	80.4	-	79.6	-
			9.5, 5.7)
	OH-16	-	-	-	3.33 brs	-	-	-	-
	17	174.6	-	175.9	-	178.6	-	175.5	-
	18	68.8	5.19 qd (6.3,	78.2	3.50 q (6.3)	78.9	3.53 q (6.3)	77.9	3.53 q (6.3)
			5.7)
	OMe-18 -	-	57.8	3.36 s	57.8	3.31 s	57.4	3.33 s
	19	18.4	1.35 d (6.3)	13.1	1.23 d (6.3)	13.6	1.19 d (6.3)	13.3	1.25 d (6.3)
	1′	173.3	-					174.1	-
	2′	34.7	2.24 t (7.6)					34	2.34 t (7.6)
	3′	25.1	1.58 m					25	1.57 m
	4′-7′	29.3-29.9 1.15-1.35 m					29.3-29.9 1.15-1.35 m
	8′	29.3-29.9 1.15-1.35 m					32.1	1.15-1.35 m
	9′	29.3-29.9 1.15-1.35 m					22.9	1.15-1.35 m
	10′	29.3-29.9 1.15-1.35 m					14.4	0.85 t (6.9)
	11′	29.3-29.9 1.15-1.35 m
	12′	29.3-29.9 1.15-1.35 m
	13′	29.3-29.9 1.15-1.35 m
	14′	32.1	1.15-1.35 m
	15′	22.9	1.15-1.35 m
	16′	14.3	0.85 t (6.9)

a Overlapped.

Table 4 . 1 H and 13 C NMR Data for Compound 14 in CDCl3

 4

			14
	position	δC	δH (J in Hz)
	1	52.8	-
	2	74.1	4.33 brd (4.0)
	OH-2	-	3.38 brd (5.1)
	3	82.6	4.86 brd (4.8)
	4	49.8	3.21 m
	5	48.9	3.12 d (4.3)
	6	77.1	-
	OH-6	-	2.52 s
	7	23.7	1.35 s
	8	141	-
	9-a	112.1	4.70 brs
	9-b		4.93 brs
	10	23.1	1.89 s
	11	61.9	3.74 d (2.8)
	12	63.2	3.34 d (2.8)
	13	90.2	-
	14-a	32.6	4.18 brd (17.8)
	14-b		2.67 brd (17.8)
	15	174.8	-
	16	128.2	-
	OH-16	-	-
	17	170.3	-
	18	136	6.69 qdd (7.1, 3.3,
			2.9)
	OMe-18 -	-
	19	16	1.83 brd (7.1)

a Overlapped.

2S,3R,4R,5S,6R,11S,12R,13S,16S,18S)-Austrobuxusin G (5): white

  , Supporting Information; HRESIMS m/z 277.1107 [M-H2O+H] + (calcd for C15H17O5 + , 277.1081). X-ray crystallographic data, see S2, Supporting Information. MeOH); 1 H and13 C NMR, see Table2; HRESIMS m/z 535.2908 [M+H] + (calcd for C29H43O9 + , 535.2913). X-ray crystallographic data, see S2, Supporting Information.

	(1R,2S,3R,4R,5S,6R,11S,12R,13S,16R,18S)-16-epi-Austrobuxusin H (7): white needles;
	GNPS ID: CCMSLIB00005723341 [M + H] + . [𝛼] $ %& -17 (c 0.1, GNPS ID: CCMSLIB00005723351 [M+H] + , CCMSLIB00005723352 [M+Na] + .
	(1R,2S,3R,4R,5S,6R,11S,12R,13R)-Austrobuxusin E (2): white amorphous powder; (1R,2S,3R,4R,5S,6R,11S,12R,13S,16R,18S)-16-epi-Austrobuxusin G (8): white
	[𝛼] $ %& +11 (c 0.1, MeOH); 1 H and 13 C NMR, see Table 1; HRESIMS m/z 477.2828 [M+H] + amorphous powder; [𝛼] $ %& -23 (c 0.1, MeOH); 1 H and 13 C NMR, see Table 2; HRESIMS m/z
	(calcd for C27H41O7 + , 477.2847). GNPS ID: CCMSLIB00005723342 [M + H] + , 563.3218 [M+H] + (calcd for C31H47O9 + , 563.3215). GNPS ID: CCMSLIB00005723353 [M+H] + ,
	CCMSLIB00005723343 [2M + H] + . CCMSLIB00005723354 [M+Na] + .
	(1R,2S,3R,4R,5S,6R,11S,12R,13S,16S,18S)-16-epi-Pretoxin (3): white needles; mp 208-(1R,2S,3R,4R,5S,6R,11S,12R,13S,16R,18S)-Austrobuxusin I (9): white crystalline
	210°C; [𝛼] $ %& +18 (c 0.1, MeOH); 1 H and 13 C NMR, see Table 1; HRESIMS m/z 381.1550 powder; mp 131-133°C; [𝛼] $ %& -24 (c 0.1, MeOH); 1 H and 13 C NMR, see Table 2; HRESIMS
	[M+H] + (calcd for C19H25O8 + , 381.1555). X-ray crystallographic data, see S2, Supporting m/z 591.3533 [M+H] + (calcd for C33H51O9 + , 591.3539). X-ray crystallographic data, see S2,
	Information. GNPS ID: CCMSLIB00005723344 [M+H] + . Supporting Information. GNPS ID: CCMSLIB00005723355	[M+H] + ,
	(1R,2S,3R,4R,5S,6R,11S,12R,13S,16S,18S)-Austrobuxusin F (4): white amorphous CCMSLIB00005723356 [M+Na] + .
	powder; [𝛼] $ %& +12 (c 0.1, MeOH); 1 H and 13 C NMR, see Table 1; HRESIMS m/z 563.3176 (1R,2S,3R,4R,5S,6R,11S,12R,13S,16R,18S)-Austrobuxusin J (10): white amorphous
	[M+H] + (calcd for C31H47O9 + , 563.3215). GNPS ID: CCMSLIB00005723345 [M+H] + , powder; [𝛼] $ %& -18 (c 0.1, MeOH); 1 H and 13 C NMR, see Table 3; HRESIMS m/z 619.3870
	CCMSLIB00005723346 [M+Na] + . [M+H] + (calcd for C35H55O9 + , 619.3852). GNPS ID: CCMSLIB00005723357 [M+H] + ,
	(1R,amorphous CCMSLIB00005723358 [M+Na] + .
	powder; [𝛼] $ %& +6 (c 0.1, MeOH); 1 H and 13 C NMR, see Table 1; HRESIMS m/z 563.3223 (1R,
	[M+H] + (calcd for C31H47O9 + , 563.3215). GNPS ID: CCMSLIB00005723347 [M+H] + ,
	CCMSLIB00005723348 [M+Na] + .
	(1R,2S,3R,4R,5S,6R,11S,12R,13S,16S,18S)-Austrobuxusin H (6): white amorphous
	powder; [𝛼] $ %& +11 (c 0.5, MeOH); 1 H and 13 C NMR, see Table 2; HRESIMS m/z 535.2928
	[M+H] + (calcd for C29H43O9 + , 535.2913). GNPS ID: CCMSLIB00005723349 [M+H] + ,
	CCMSLIB00005723350 [M+Na] + .

2S,3R,4R,5S,6R,11S,12R,13S,16S,18S)-16-epi-Austrobuxusin B (11): white needles;

	(1R,2S,3R,4R,5S,6R,11S,12R,13S,16S,18S)-Austrobuxusin L (13): white amorphous
	powder; [𝛼] $ %& 0 (c 0.1, MeOH); 1 H and 13 C NMR, see Table 3; HRESIMS m/z 567.3195
	[M+H] + (calcd for C30H47O10 + , 567.3175). X-ray crystallographic data, see S2, Supporting
	Information.
	(1R,2S,3R,4R,5S,6R,11S,12R,13S)-Austrobuxusin M (14): white needles; [𝛼] $ %& +15 (c
	0.1, MeOH); 1 H and 13 C NMR, see Table 4; HRESIMS m/z 363.1467 [M+H] + (calcd for
	C19H23O7 + , 363.1449). X-ray crystallographic data, see S2, Supporting Information.
	X-ray Crystallography. X-ray analyses experimental details, ORTEP views, Crystal data,
	Structure refinement and Conventional hydrogen bonds of compounds 1, 3, 7, 9, 11, 12, 13
	and 14 are available in S2 Supporting Information. Crystallographic data have been deposited
	in the Cambridge Crystallographic Data Center as entries 1996118-1996126 (compounds 1,
	3, 7, 9, 11, 12, 13, and 14 (both polymorphs) respectively) and can be obtained, free of
	charge, on application to the Director, CCDC, 12 Union Rd., Cambridge CB21EZ, U.K. [fax,
	+44(0)-1233-336033; email, deposit@ccdc.cam.ac.uk].
	[𝛼] $ %& -21 (c 0.1, MeOH); 1 H and 13 C NMR, see Table 3; HRESIMS m/z 411.1680 [M+H] +
	(calcd for C20H27O9 + , 411.1661). X-ray crystallographic data, see S2, Supporting Information.
	GNPS ID: CCMSLIB00005723359 [M+H] + .
	(1R,2S,3R,4R,5S,6R,11S,12R,13S,16S,18S)-Austrobuxusin K (12): white needles; [𝛼] $ %& -
	18 (c 0.1, MeOH); 1 H and 13 C NMR, see Table 3; HRESIMS m/z 413.1850 [M+H] + (calcd for
	C20H29O9 + , 413.1817). X-ray crystallographic data, see S2, Supporting Information. GNPS ID:
	CCMSLIB00005723360 [M+H] + .

Conversion of Compound 3 into Compounds 4 and 5. To

  MeCN 25:75 at 4.5 mL.min -1 ) afforded the semi-synthetic compounds 4 (5.6 mg, 10 µmol, 18%, tR 13.4 min) and 5 (0.3 mg, 0.5 µmol, 1%, tR 12.1 min). 922. MS 1 scan was followed by MS 2 scans of the five most intense ions above an absolute threshold of 3000 counts. Selected parent ions were fragmented with a collision energy fixed at 35 eV and an isolation window of 1.3 amu. The 292 MS 2 data files were converted from the .d (Agilent) standard data-format to .mzXML format using MSConvert software, part of the ProteoWizard package.

	1.05 equiv.) in anhydrous dichloromethane (2 mL) at 0 °C under an argon atmosphere, was added compound 3 (21 mg, 55 µmol, 1 equiv.). The reaction mixture was stirred at room temperature for 24 h, transferred to a separatory funnel and subsequently washed three times with an aqueous solution of NaHCO3. The organic layer was dried with MgSO4 then filtered Ultimate 3000 RSLC system equipped with an Accucore C18 column (2.1 × 100 mm; 2.6 μm, ThermoScientific). The mobile phase consisted of water-acetonitrile (H2O-CH3CN) with 0.1% formic acid 20:80 held for 5 min, then a gradient from 20:80 to 100:0 in 20 min, at a flow rate of 350 µL.min -1 . The temperature of the column oven was set at 30 °C and the injection volume at 5 μL. Use of a calibration solution, containing two internal reference masses (purine, C5H4N4, m/z 121.0509, and HP-921 [hexakis-(1H,1H,3H-tetrafluoropentoxy)phosphazene], C18H18O6N3P3F24, and concentrated in vacuo. Data Dependent LC-ESI-HRMS 2 Analysis. LC analyses were performed with a Dionex m/z 922.0098), routinely led to mass accuracy below 2 ppm. LC-UV data were analyzed with

a solution of DMAP (1 mg, 11 µmol, 0.2 equiv.), EDCI.HCl (12 mg, 60 µmol, 1.1 equiv.) and lauric acid (12 mg, 58 µmol, 1 H NMR analysis confirmed the presence of the two ester products 4 and 5 in an 8:2 ratio, respectively. Purification by semi-preparative HPLC (Kinetex C18, H2O-LC-HRESIMS 2 analysis was achieved by coupling the LC system to a hybrid quadrupole time-of-flight mass spectrometer Agilent 6540 (Agilent Technologies, Massy, France) equipped with an ESI dual source, operating in the positive-ion mode. Source parameters were set as follows: capillary temperature 325 °C, source voltage 3500 V, sheath gas flow rate 7 L.min -1 , nebulizer pressure 30 psi, drying-gas flow rate 10 L.min -1 , drying-gas temperature 350 °C, stealth-gas temperature 350 °C, skimmer voltage 45 V, fragmentor voltage 150 V, and nozzle voltage 500 V. MS scans were operated in the full-scan mode from m/z 100 to 1000 (0.1 sec scan time) with a mass resolution of 20,000 at m/z Chromeleon software (Dionex) and MS data acquisition and processing were performed using MassHunter Workstation software (Agilent Technologies, Massy, France).

MZmine 2 and Sirius Parameters.

ACKNOWLEDGMENTS

The authors are very grateful to the South Province of New Caledonia that has facilitated our field investigation and B. Fogliani for his contribution to species collection. We thank H. Lévaique and J. Bignon, who performed the cytotoxic assays on the three cancer cell lines. This work has benefited from an "Investissement d'Avenir" grant managed by Agence Nationale de la Recherche (CEBA, ref ANR-10-LABX-25-01).

compounds 1, 3, 7, 9, 11, 12, 13 and 14, 1 H, 13 C, HSQC, HMBC, COSY, ROESY NMR spectra of compounds 1-4, 6, 9, 11-14, 1 H, 13 C of compounds 5, 8, 10, 1 H, 13 C, HMBC of compound 7 and HRESIMS data for compounds 1-14. (PDF)

The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION

Notes

The authors declare no conflict of interest.