Céline Ramdani
email: celineramdani@hotmail.fr

Franck Vidal

Alain Dagher

Laurence Carbonnell

Thierry Hasbroucq

Dopamine and response selection: an Acute Phenylalanine/Tyrosine Depletion study

Keywords: Dopamine, Supplementary motor areas, Simon task, Electroencephalography, Response selection, Acute phenylalanine/tyrosine depletion: APTD

The role of dopaminergic system in decision-making is well documented, and evidence suggests that it could play a significant role in response selection processes. The N-40 is a fronto-central event-related potential, generated by the supplementary motor areas (SMAs) and a physiological index of response selection processes. The aim of the present study was to determine whether infraclinical effects of dopamine depletion on response selection processes could be evidenced via alterations of the N-40. We obtained a dopamine depletion in healthy volunteers with the acute phenylalanine and tyrosine depletion (APTD) method which consists in decreasing the availability of dopamine precursors. Subjects realized a Simon task in the APTD condition and in the control condition. When the stimulus was presented on the same side as the required response, the stimulus-response association was congruent and when the stimulus was presented on the opposite side of the required response, the stimulus-response association was incongruent. The N-40 was smaller for congruent associations than for incongruent associations. Moreover, the N-40 was sensitive to the level of dopaminergic activity with a decrease in APTD condition compared to control condition. This modulation of the N-40 by dopaminergic level could not be explained by a global decrease of cerebral electrogenesis, since negativities and positivities indexing the recruitment of the primary motor cortex (anatomically adjacent to the SMA) were unaffected by APTD. The specific sensitivity of N-40 to ATPD supports the model of Keeler et al. (Neuroscience 282:156-175, 2014) according to which the dopaminergic system is involved in response selection.

Introduction

Decision-making can be regarded as a set of cognitive processes that contribute to the production of the optimal alternative among a set of concurrently possible actions. The role of the dopaminergic system in human decision-making is well documented (e.g., [START_REF] Montague | A framework for mesencephalic dopamine systems based on predictive Hebbian learning[END_REF][START_REF] Montague | Computational roles for dopamine in behavioural control[END_REF][START_REF] Rogers | The roles of dopamine and serotonin in decision making: evidence from pharmacological experiments in humans[END_REF].

Response selection (i.e., the association of a specific action to a specific sensation) can be considered as the core process of decision-making [START_REF] Keeler | Functional implications of dopamine D1 vs. D2 receptors: a 'prepare and select' model of the striatal direct vs. indirect pathways[END_REF], and one can wonder whether the dopaminergic system is directly involved in this process. According to [START_REF] Keeler | Functional implications of dopamine D1 vs. D2 receptors: a 'prepare and select' model of the striatal direct vs. indirect pathways[END_REF], the striatal direct pathway (D1 receptors subtype) would allow preparation for response selection, while the striatal indirect pathway (D2 receptor subtypes) would allow selection of the appropriate response within the prepared set of all possible responses, through. [START_REF] Keeler | Functional implications of dopamine D1 vs. D2 receptors: a 'prepare and select' model of the striatal direct vs. indirect pathways[END_REF] called this system a Bprepare and selectâ rchitecture.

The implication of the dopaminergic system in preparatory processes has been widely acknowledged in animals. Response preparation is impaired in rats after dopamine depletion [START_REF] Brown | Simple and choice reaction time performance following unilateral striatal dopamine depletion in the rat[END_REF]. Now, [START_REF] Yoon | Delay period activity of the substantia nigra during proactive control of response selection as determined by a novel fMRI localization method[END_REF] went a step further showing that, during preparation, the activation of the dopaminergic system adjusts to the difficulty of the response selection to be performed after this preparation.

In humans, taking advantage of the high iron concentration in the substantia nigra (SN) which reveals this structure as a relatively hypodense zone on T2*-weighted images (including EPI volumes), [START_REF] Yoon | Delay period activity of the substantia nigra during proactive control of response selection as determined by a novel fMRI localization method[END_REF] accurately examined the activation of the SN during the 7.5-s preparatory period of a between-hand choice reaction time (RT) task. At the beginning of the preparatory period, a precue indicated which one of two (easy or difficult) stimulus-response associations should be applied when the response signal (RS) would be delivered, at the end of the preparatory period. The SN BOLD signal increased after the precue in both cases. However, whereas the BOLD signal returned to baseline towards the end of the preparatory period in the easiest of the two possible response selection conditions, this signal remained at high levels until the end of the preparatory period in the most difficult condition. Interestingly, no such an interaction could be evidenced in the neighboring subthalamic nucleus (STN); given the close functional relationships between STN and SN pars reticulata, the authors convincingly argued that the BOLD signal sensitivity to the difficulty of the selection process resulted from a sensitivity of the dopaminergic neurons of SN pars compacta to this manipulation. This interpretation is highly consistent with Keeler et al.'s (2014) model which assumes that the dopaminergic system plays a prominent role in response selection processes. Now, after the RS, that is in the period when response selection itself occurs, no differential effect could be evidenced, but this might easily be explained by the poor temporal resolution of fMRI method (RTs were about 550 and 600 ms only, in the easy and difficult conditions, respectively).

Evidencing a direct effect of the dopaminergic system on response selection processes themselves (which take place during the RT period) would lend direct additional support to Keeler et al.'s view that the dopaminergic system plays an essential role in response selection, not only in preparing for its difficulty [START_REF] Yoon | Delay period activity of the substantia nigra during proactive control of response selection as determined by a novel fMRI localization method[END_REF] but also in carrying out response selection processes themselves; this was the aim of the present study which will be made explicit as follows.

Among the main targets of the basal ganglia (via the thalamus) are the supplementary motor areas (SMAs). Recent fMRI data did demonstrate that acute diet-induced dopamine depletion (APTD) impairs timing in humans by decreasing activity not only in the putamen but also in the SMAs [START_REF] Coull | Dopamine precursor depletion impairs timing in healthy volunteers by attenuating activity in putamen and supplementary motor area[END_REF], which role in motor as well as sensory timing is well documented (e.g., [START_REF] Coull | Neuroanatomical and neurochemical substrates of timing[END_REF].

SMAs are often assumed to play a prominent role not only in timing but also in response selection (e.g., Mostofsky and Simmonds 2008, for a review). Taking into account the sensitivity to dopamine depletion of the SMAs [START_REF] Coull | Dopamine precursor depletion impairs timing in healthy volunteers by attenuating activity in putamen and supplementary motor area[END_REF], one might therefore wonder, in the frame of Keeler et al.'s (2014) model, whether their activities would also be impaired, by APTD during the reaction period of a RT task in which a response selection is required. Given the short time range of RTs, a high temporal resolution method is needed to address this question. EEG seems to be particularly adapted, since it is classically considered as having an excellent temporal resolution [START_REF] Sejnowski | Brain and cognition[END_REF].

During the reaction time of a between-hand choice RT task, an electroencephalographic (EEG) component has been evidenced in humans (the N-40; [START_REF] Vidal | An ERP study of cognitive architecture and the insertion of mental processes: Donders revisited[END_REF] right over the SMAs. Given that the N-40 peaks about 50 ms before the peak activation of the (contralateral) primary motor cortex involved in the response, it has been proposed that this component is an index of response selection which might arise from the SMAs. In accordance to this view, it has been shown that (1) that the N-40, although present in choice conditions was absent in a go/ no-go task, a task in which no response selection is required [START_REF] Vidal | An ERP study of cognitive architecture and the insertion of mental processes: Donders revisited[END_REF]and (2) that the amplitude of the N-40 was modulated by the difficulty of the selection process, being smaller for easier selections [START_REF] Carbonnell | The N-40: an electrophysiological marker of response selection[END_REF]. Finally, tentative source localization performed with two independent methods (sLORETA and BESA), pointed to quite superficial medio frontal generators corresponding to the SMAs [START_REF] Carbonnell | The N-40: an electrophysiological marker of response selection[END_REF]).

If we admit that the N-40 is generated by the SMAs and index response selection, a convenient way to address the question of the involvement of the dopaminergic system in response selection processes consists in examining the sensitivity of the N-40 to APTD in a between-hand choice RT task quite similar to the one used by [START_REF] Yoon | Delay period activity of the substantia nigra during proactive control of response selection as determined by a novel fMRI localization method[END_REF]: we chose a Simon task (see Fig. 1, see [START_REF] Simon | The effects of an irrelevant directional cue on human information processing[END_REF] for a review). Now, in between-hand choice RT tasks, the N-40 is followed by a transient (negative) motor potential [START_REF] Deecke | Voluntary finger movement in man: cerebral potentials and theory[END_REF]) revealing the build-up of the motor command in the (contralateral) primary motor areas (M1) controlling the responding hand [START_REF] Arezzo | Intracortical sources and surface topography of the motor potential and somatosensory evoked potential in the monkey[END_REF]. Concurrently, a transient positive wave, reflecting motor inhibition and related to error prevention [START_REF] Meckler | Motor inhibition and response expectancy: a Laplacian ERP study[END_REF], develops over (ipsilateral) M1 controlling the non-responding hand [START_REF] Vidal | An ERP study of cognitive architecture and the insertion of mental processes: Donders revisited[END_REF]. If one admits that these activities are not directly related to response selection, examining their (in)sensitivity to dopamine depletion allows examining the selectivity of the effects of this depletion (if present) on response selection processes.

In a previous study, we submitted subjects to APTD. Although we did evidence subtle behavioral effects that can be attributed to action monitoring impairments [START_REF] Ramdani | Dopamine precursors depletion impairs impulse control in healthy volunteers[END_REF], we did not find any clear behavioral evidence of APTD-induced response selection impairment.

To evidence the role of the dopaminergic system in response selection, the present study was aimed at assessing whether infraclinical effects of dopamine depletion on response selection processes can be evidenced via selective alterations of the N-40.

Material and method

The experimental procedure has been described in detail elsewhere [START_REF] Ramdani | Dopamine precursors depletion impairs impulse control in healthy volunteers[END_REF], and only essential information is provided here.

Twelve healthy subjects participated in this experiment.

Dopamine depletion

Dopamine availability was decreased using the APTD method [START_REF] Mctavish | Effect of a tyrosine-free amino acid mixture on regional brain catecholamine synthesis and release[END_REF][START_REF] Leyton | Effects on mood of acute phenylalanine/tyrosine depletion in healthy women[END_REF][START_REF] Leyton | Decreasing amphetamine-induced dopamine release by acute phenylalanine/tyrosine depletion: a PET/ [ 11 C] raclopride study in healthy men[END_REF][START_REF] Nagano-Saito | Dopamine depletion impairs frontostriatal functional connectivity during a set-shifting task[END_REF], 2012). The present experiment comprised two experimental sessions differing by the level of tyrosine and phenylalanine in the amino acid mixture: (i) the Bplacebo session^: the subject performed the task after ingestion of a mixture containing 16 essential amino acids (including tyrosine and phenylalanine) and (ii) the Bdepleted session^: the subject performed the task after the ingestion of the mixture without tyrosine and phenylalanine. Plasma concentrations of phenylalanine, tyrosine, and other large neutral amino acids (LNAAs; leucine, isoleucine, methionine, valine, and tryptophane) were measured by HPLC with fluorometric detection on an Ultrasphere ODS reversephase column (Beckman Coulter) with ophtalaldehyde precolumn derivatization and amino-adipic acid as an internal standard. Plasma concentrations of tryptophan were measured by HPLC-FD on a Bondpak reverse-phase column (Phenomenex).

Task and design

Each subject performed both sessions, on separate days, at least 3 days apart. Subjects were not taking any medication at the time of the experiment. None of them had a history of mental or neurologic illness. They were asked not to take stimulating substances (e.g., caffeine or stimulant drugs) or alcohol the day and the night before both sessions. The day before each session, subjects ate a low protein diet provided by investigators and fasted after midnight. On the test days, subjects arrived at 8:30 a.m. at the laboratory and had blood sample drawn to measure plasma amino acid concentrations. They ingested one of the two amino acid mixtures at 9:00 a.m. in a randomized, double-blind manner. Peak dopamine reduction occurs during a period 4-6 h after ingestion of the two amino acid mixtures [START_REF] Leyton | Decreasing amphetamine-induced dopamine release by acute phenylalanine/tyrosine depletion: a PET/ [ 11 C] raclopride study in healthy men[END_REF]). They were tested from 1:30 p.m. At 3:00 p.m., subjects had a second blood sample drawn to measure plasma amino acid concentrations.

The order of depleted and placebo sessions was counterbalanced between subjects.

Subjects performed a between-hand choice reaction time RT task.

A trial began with the presentation of a stimulus. The subjects' responses turned off the stimulus, and 500 ms later, the next stimulus was presented. If subjects had not responded within 800 ms after stimulus onset, the stimulus was turned off and the next stimulus was displayed 500 ms later.

At the beginning of an experimental session, subjects had one training block of 129 trials. Then, they were required to complete 16 blocks of 129 trials each. A block lasted about 2 min. There was 1 min break between two blocks and 5 min break every four blocks. The training block was discarded from statistical analyses.

The structure of this between-hand choice RT task realized a Simon task [START_REF] Simon | The effects of an irrelevant directional cue on human information processing[END_REF]. The stimuli of this Simon task were the digits three, four, six, and seven presented either to the right or the left of a central fixation point. Half of the subjects responded with the right thumb on the right force sensor for even digits and with the left thumb on the left force sensor for odd digits; the other half performed the reverse mapping. When the stimulus was presented on the same side as the required response, the stimulus-response association was congruent. When the stimulus was presented on the side opposite to the required response, the stimulus-response association was incongruent. A block contained 50% of the congruent trials and 50% of the incongruent ones. [START_REF] Yoon | Delay period activity of the substantia nigra during proactive control of response selection as determined by a novel fMRI localization method[END_REF] (see Fig. 1) manipulated the complexity of the stimulus-response association by varying the spatial correspondence between the direction (right or left) indicated by a centrally presented arrow and the position (right or left) of the required response: on congruent associations, responses had to be given on the side indicated by the arrow, while on incongruent associations, responses had to be given on the opposite side. In the present Simon task, congruency was manipulated by varying the spatial correspondence between the position of the stimulus and the position of the required response, given that (1) as in the [START_REF] Yoon | Delay period activity of the substantia nigra during proactive control of response selection as determined by a novel fMRI localization method[END_REF] study, congruency affects response selection processes [START_REF] Hommel | A feature-integration account of sequential effects in the Simon task[END_REF][START_REF] Kornblum | The effects of irrelevant stimuli: the time course of S-S and S-R consistency effects with Stroop-like stimuli, Simon-like tasks, and their factorial combinations[END_REF]Proctor and Reeve 1990) and (2) congruency affects the amplitude of the N-40 in a Simon task [START_REF] Carbonnell | The N-40: an electrophysiological marker of response selection[END_REF]. Subjects were asked to respond as fast and as accurately as possible.

RT was defined by the time interval between the stimulus onset and the mechanical response.

Electrophysiological recordings and processing

The electromyographic (EMG) activity of the flexor pollicis brevis (thenar eminence, inside base of the thumb) was recorded bipolarly by means of surface Ag-AgCl electrodes, 6 mm in diameter, fixed about 20 mm apart on the skin of each thenar eminence. The recorded EMG signals were digitized online (bandwidth 0-268 Hz, 3 db/octave, sampling rate 1024 Hz), filtered off-line (high pass = 10 Hz), and then, inspected visually [START_REF] Van Boxtel | Detection of EMG onset in ERP research[END_REF]). The EMG onsets were hand scored because human pattern recognition processes are superior to automated algorithms (see [START_REF] Staude | Precise onset detection of human motor responses using a whitening filter and the log-likelihood-ratio test[END_REF]). To overcome subjective influence on the scoring, the experimenter who processed the signals was unaware of the type of associations (congruent, incongruent) or session (placebo, APTD) to which the traces corresponded.

Electroencephalogram (EEG) and electro-oculogram (EOG) were recorded continuously from preamplified Ag/ AgCl electrodes (BIOSEMI Active-Two electrodes, Amsterdam). For EEG, 64 recording electrodes were disposed according to the 10/20 system with CMS-DRL as reference and ground (specific to the Biosemi acquisition system). A 65th electrode on the left mastoid served to reference the signal offline. Electrodes for vertical and horizontal EOG were at the Fp1 and below the left eye and at the outer canthus of the left and right eyes, respectively. The signal was filtered and digitized online (bandwidth 0-268 Hz, 3 db/octave, sampling rate 1024 Hz). EEG and EOG data were numerically filtered offline (high pass = 0.02 Hz). No additional filtering was performed. Bipolar derivations were calculated offline for vertical and horizontal EOGs. Then, ocular artifacts were subtracted [START_REF] Gratton | A new method for off-line removal of ocular artifact[END_REF]. A trial-by-trial visual inspection of monopolar recordings allowed us to reject unsatisfactory subtractions and other artifacts.

The scalp potential data were segmented from (-500) to (+ 500 ms) with the EMG onset as zero of time. Afterward, for each individual, scalp potential data were averaged time locked to the EMG onset. However, on scalp potential data, due to volume conduction effects, the N-40 is overlapped by large components generated by remote generators and by closer ones in the primary motor areas. Therefore, it hardly shows up on scalp potential recordings [START_REF] Vidal | An ERP study of cognitive architecture and the insertion of mental processes: Donders revisited[END_REF].

The surface Laplacian (SL) transformation (see Carvalhaes and de [START_REF] Carvalhaes | The surface Laplacian technique in EEG: theory and methods[END_REF] for theory and methods), acting as a high-pass spatial filter [START_REF] Nuñez | Estimation of large scale neocortical source activity with EEG surface Laplacians[END_REF], is very efficient in attenuating volume conduction effects [START_REF] Giard | Scalp current density mapping in the analysis of mismatch negativity paradigms[END_REF][START_REF] Kayser | On the benefits of using surface Laplacian (current source density) methodology in electrophysiology[END_REF]. Because of this property, the SL transformation allows unmasking the N-40, by removing spatial overlap between this component and other ones. Therefore, the Laplacian transformation was applied on each individual scalp potential average, obtained for each subjects in each condition (congruent and incongruent), and each session (placebo and APTD); surface Laplacian was estimated after spherical spline interpolation with four as the degree of spline and a maximum of 10°for the Legendre polynomial, according to the method of [START_REF] Perrin | Scalp current density mapping: value and estimation from potential data[END_REF].

N-40 At the FCz electrode, the N-40 begins to develop about 70 ms and peaks about 20 ms before EMG onset. The slopes of the linear regression of this wave were computed for each subject from -70 to -20 ms, so within a 50-ms time window. The slopes of the N-40 were determined for Bpure^correct trials only. These values were then submitted to repeatedmeasures canonical analyses of variance (ANOVA). The ANOVA involved two within-subjects factors: session (placebo, APTD) and congruency (congruent, incongruent) for mean results. Contrary to stimulus-locked data, when studying response-locked averages, the choice of the appropriate baseline is always problematic. To circumvent the problem, peakto-peak measures are often used, as they are baseline free.

They may be conceived of as a crude slope measure. However, mean slopes can be estimated by computing the linear regression line by the least squares method, in an interval of interest. In this case, slope analysis has certain advantages over amplitude analysis: (i) they are also independent of the baseline and (ii) they give morphological information on the polarity of the curves in delimited time windows and are less variable than amplitude measures [START_REF] Meckler | Motor inhibition and response expectancy: a Laplacian ERP study[END_REF].

Activation/inhibition pattern For correct trials, we analyzed EEG activities over the primary sensory-motor area (SM1) contralateral and ipsilateral to the response (over C3 and C4 electrodes): we measured the slopes computed by linear regression in a specific time window of 50 ms (-30 ms; 20 ms) (e.g., [START_REF] Meckler | Motor inhibition and response expectancy: a Laplacian ERP study[END_REF]. These mean values of slope were submitted to an ANOVA involved two within-subjects factors: session (placebo, APTD) and congruency (congruent, incongruent).

Results

Amino acid plasmatic concentrations

For phenylalanine (see Table 1), the ANOVA revealed an effect of the session (F (1, 11) = 37.49; p = 0.000075) and no effect of the time of the drawn (F (1, 11) = 0.13; p = 0.73). These two factors interacted (F (1, 11) = 119.81; p = 0.000000), signaling that the session affected the samples drawn at the end of the testing session, 6 h after ingestion (F (1, 11) = 65.84; p = 0.000006), but not the samples drawn before ingestion (F (1, 11) = 0.016; p = 0.902). For tyrosine (see Table 1), the ANOVA revealed a main effect of the session (F (1, 11) = 68.91; p = 0.000005) and a main effect of time (F (1, 11) = 17.95; p = 0.0014). These two factors interacted (F (1, 11) = 68.83; p = 0.000005), indicating that the session affected tyrosine levels at the end of the session (F (1, 11) = 71.16; p = 0.000004) but not prior to ingestion (F (1, 11) = 0.24; p = 0.631).

In sum, plasma concentrations of tyrosine and phenylalanine were significantly lower for the depleted session than for the placebo session.

Reaction time of correct responses

There was a main effect of congruency of 13 ms (congruent 408 ms, incongruent 421 ms, F (1, 11) = 57.58; p = 0.00001) but no effect of session (placebo 414 ms; depleted 416 ms, F (1, 11) = 0.056; p = 0.817). These two factors did not interact on mean RT (F (1, 11) = 0.116; p = 0.739).

Error rate

There was a non-significant trend for an increase of error rate on incongruent stimulus-response associations (7.28%) compared to congruent stimulus-response associations (5.79%) (F = 3.70; p = 0.081). The error rate was not statistically different for the placebo (6.62%) and depleted (6.45%) sessions (F (1, 11) = 0.123; p = 0.732). There was no interaction between congruency and session (F (1, 11) = 0.026; p = 0.874).

N-40 (Fig. 2)

Only data obtained on correct trials have been analyzed.

As expected from [START_REF] Carbonnell | The N-40: an electrophysiological marker of response selection[END_REF], the slope of the N-40 was steeper on the incongruent (-57.42 μV/cm 2 /ms) than on the congruent condition (-24.50 μV/cm 2 /ms) (size effect on steepness -32.92 μV/cm 2 /ms, F (1, 11) = 5.41, p = 0.040). The congruency effect observed on slopes of the N-40 was attributed to the more demanding selection on incongruent stimulus-response associations as compared to congruent stimulus-response associations. Moreover, the slope of the N-40 was also steeper on placebo (-59.39 μV/cm 2 /ms) than on the APTD session (-22.54 μV/cm 2 /ms) (size effect on steepness -36.85 μV/cm 2 /ms, F (1, 11) = 5.50, p = 0.039).

These two factors (congruency and sessions) did not interact (F (1, 11) = 0.030; p = 0.865).

Activation/inhibition pattern (Fig. 3) Inspection of the Laplacian traces reveals that, for all conditions, a negativity/positivity pattern developed before EMG onset over the controlateral and ipsilateral M1s respectively.

As expected from [START_REF] Vidal | An ERP study of cognitive architecture and the insertion of mental processes: Donders revisited[END_REF], over contralateral M1 (over controlateral electrode), we observed a negative wave peaking about EMG onset while over the ipsilateral M1 (over ipsilateral electrode), we observed a positive wave.

Regarding contralateral negativity, there was no effect of congruency (congruent condition -210.37 μV/cm 2 /ms and incongruent condition -224.19 μV/cm 2 /ms; F (1, 11) = 0.67; p = 0.429) or main effect of the session (placebo session -213.10 μV/cm 2 /ms, APTD session -221.47 μV/cm 2 /ms; F (1, 11) = 0.066; p = 0.802). These two factors did not interact (F (1, 11) = 1.18, p = 0.299).

Regarding ipsilateral positivity, there was neither an effect of congruency (congruent condition 102.51 μV/ms and incongruent condition 110.26 μV/ms; F (1, 11) = 0.349; p = 0.566) nor an effect of session (placebo session 109.53 μV/ms, APTD session 103.25 μV/ms; F (1, 11) = 0.182; p = 0.677). These two factors did not interact (F (1, 11) = 0.098; p = 0.759).

Discussion

The present study reproduces already available data: (1) RTs were longer on congruent than on incongruent trials, revealing the existence of a Simon effect; (2) an activation/inhibition pattern developed over M1s before the response [START_REF] Vidal | An ERP study of cognitive architecture and the insertion of mental processes: Donders revisited[END_REF][START_REF] Van De Laar | Lifespan changes in motor activation and inhibition during choice reactions: a Laplacian ERP study[END_REF][START_REF] Alexander | Linking motor-related brain potentials and velocity profiles in multi-joint arm reaching movements[END_REF]); (3) this activation/inhibition pattern was preceded by an N-40 [START_REF] Vidal | An ERP study of cognitive architecture and the insertion of mental processes: Donders revisited[END_REF][START_REF] Alexander | Linking motor-related brain potentials and velocity profiles in multi-joint arm reaching movements[END_REF], which is in line with the notion that N-40 index response selection processes, upstream to response execution as manifested by contralateral M1 activation; (4) the amplitude of the N-40 depended on congruency, being larger on congruent than on incongruent associations [START_REF] Carbonnell | The N-40: an electrophysiological marker of response selection[END_REF], which is also in line with the notion that N-40 indexes response selection processes; (5) the procedure used here was efficient in inducing a clear ATPD, known to induce a secondary dopamine depletion (McTavish et al. [START_REF] Leyton | Effects on mood of acute phenylalanine/tyrosine depletion in healthy women[END_REF][START_REF] Leyton | Decreasing amphetamine-induced dopamine release by acute phenylalanine/tyrosine depletion: a PET/ [ 11 C] raclopride study in healthy men[END_REF][START_REF] Nagano-Saito | Dopamine depletion impairs frontostriatal functional connectivity during a set-shifting task[END_REF], 2012); (6) acute dopamine depletion had no effect on RT, error rate, or the size of the congruency effect, in line with Larson et al.'s (2015) results who did not evidence either any APTD effect on RT, error rates, or the size of the congruency effect, in another conflict task. Therefore, given that the procedure used here induced a clear APTD, and that subjects exhibited behavioral and EEG patterns as expected from previous literature, we can be quite confident that subjects were submitted to classical appropriate experimental conditions.

In these conditions, (1) over M1s, neither contralateral activation nor ipsilateral inhibition were sensitive to congruency, suggesting that congruency has little or no effect on execution processes (contralateral M1) or error prevention (ipsilateral M1); (2) over M1s, neither contralateral activation nor ipsilateral inhibition were sensitive to APTD, suggesting that the dopamine depletion has little or no effect on execution processes (contralateral M1) or error prevention (ipsilateral M1);

(3) over the SMAs, the N-40 was reduced after APTD, suggesting that the dopamine depletion affects response selection processes.

A first comment is in order. The sensitivity of the N-40 to ATPD cannot be attributed to a general effect on cerebral electrogenesis. First, because ERPs recorded over M1s were unaffected by ATPD; secondly, because Larson and his colleagues (2015) examined extensively the sensitivity to ATPD of several ERPs, assumed to reveal action monitoring processes, namely the N450 [START_REF] West | Effects of task context and fluctuations of attention on neural activity supporting performance of the Stroop task[END_REF], the conflict slow potential [START_REF] West | Effects of task context and fluctuations of attention on neural activity supporting performance of the Stroop task[END_REF][START_REF] Mcneely | Neurophysiological evidence for disturbances of conflict processing in patients with schizophrenia[END_REF], the Error Negativity [START_REF] Falkenstein | Effects of crossmodal divided attention on late ERP components. II. Error processing in choice reaction tasks[END_REF][START_REF] Gehring | A neural system for error detection and compensation[END_REF], or the Error Positivity [START_REF] Falkenstein | Effects of crossmodal divided attention on late ERP components. II. Error processing in choice reaction tasks[END_REF], and none of these activities were sensitive to ATPD, thus confirming that the present effects observed on the N-40 cannot result from a general decrease of electrogenesis. Therefore, one can conclude that the sensitivity of the N-40 to ATPD is specific.

Second, because dopamine depletion has little or no effect on execution processes (contralateral M1) or proactive control of errors (ipsilateral M1), one can conclude that the effect of ATPD observed on the N-40 over the SMAs reflects a selective influence of dopamine depletion on response selection processes (as proposed in the BIntroduction^section), without noticeable effects on processes occurring downstream, i.e., Fig. 2 N-40: in black, the placebo session and in gray, the APTD session; in dash, the congruent condition and in solid the incongruent condition. Maps have the same scale and dated at -30 ms. The zero of time corresponds to the onset of the EMG burst response execution. Note that the selective influence of APTD, but also of congruency, on upstream processes, both leaving unaffected contingent downstream activities, suggests the existence of separate modules in information processing operations [START_REF] Sternberg | Separate modifiability, mental modules, and the use of pure and composite measures to reveal them[END_REF][START_REF] Sternberg | Modular processes in mind and brain[END_REF] and the existence of Bfunctionally specialized [neural] processing modules ( Sternberg 2011, page 158). Now, because APTD had no influence on RT, error rate, or the size of the congruency effect [START_REF] Ramdani | Dopamine precursors depletion impairs impulse control in healthy volunteers[END_REF][START_REF] Larson | The effects of acute dopamine precursor depletion on the cognitive control functions of performance monitoring and conflict processing: an event-related potential (ERP) study[END_REF], we must conclude that the effect of an about 30% dopamine depletion [START_REF] Leyton | Decreasing amphetamine-induced dopamine release by acute phenylalanine/tyrosine depletion: a PET/ [ 11 C] raclopride study in healthy men[END_REF][START_REF] Montgomery | Reduction of brain dopamine concentration with dietary tyrosine plus phenylalanine depletion: an [11C] raclopride PET study[END_REF]) observed here on the N-40 reveals a weak infraclinical functional deficit in response selection operations. One can imagine that a stronger depletion would have a behavioral expression on RTs.

According to the model of [START_REF] Keeler | Functional implications of dopamine D1 vs. D2 receptors: a 'prepare and select' model of the striatal direct vs. indirect pathways[END_REF], fMRI data reported by [START_REF] Yoon | Delay period activity of the substantia nigra during proactive control of response selection as determined by a novel fMRI localization method[END_REF] indicate that during the preparatory period of a choice RT task, the dopaminergic system is involved in preparing for response selection; the SN pars compacta BOLD signal increased after the precue but returned to baseline before the end of the preparatory period in the easiest of the two possible response selection conditions. However due to the low temporal resolution of fMRI, no evidence could be provided regarding response selection per se.

If one admits that the N-40 is a physiological index of response selection processes [START_REF] Vidal | An ERP study of cognitive architecture and the insertion of mental processes: Donders revisited[END_REF][START_REF] Carbonnell | The N-40: an electrophysiological marker of response selection[END_REF], the selective sensitivity of the N-40 to ATPD, with spared M1s activation/inhibition pattern, lends support to the model of [START_REF] Keeler | Functional implications of dopamine D1 vs. D2 receptors: a 'prepare and select' model of the striatal direct vs. indirect pathways[END_REF] which assumes that the dopaminergic system is involved in response selection per se. In the motor loop between the basal ganglia and the cortex [START_REF] Alexander | Linking motor-related brain potentials and velocity profiles in multi-joint arm reaching movements[END_REF], the SMAs constitute a major cortical target of the basal ganglia, via the thalamus. If we assume that the N-40 is generated by the SMAs [START_REF] Carbonnell | The N-40: an electrophysiological marker of response selection[END_REF], it is likely that the final effect of dopaminergic depletion on response selection takes place in the SMAs because of a decrease in thalamic glutamatergic output to this area, due to dopamine depletion-induced striatal impairment. Of course, it cannot be excluded that APTD influenced SMAs activity through direct dopaminergic projections to the cortex; however, this seems unlikely since PET studies show that most of the APTD-induced dopamine depletion involves striatal structures [START_REF] Leyton | Decreasing amphetamine-induced dopamine release by acute phenylalanine/tyrosine depletion: a PET/ [ 11 C] raclopride study in healthy men[END_REF][START_REF] Montgomery | Reduction of brain dopamine concentration with dietary tyrosine plus phenylalanine depletion: an [11C] raclopride PET study[END_REF].

Fig. 3 Activation/inhibition pattern: In green, the placebo session; light green: activation of the (contralateral) primary motor cortex involved in the response; and dark green: inhibition of the (contralateral) primary motor cortex involved in the response. In red, the APTD session; dark red: activation of the (contralateral) primary motor cortex involved in the response; and light red: inhibition of the (ipsilateral) primary motor cortex involved in the response. The zero of time corresponds to the onset of the EMG burst. Maps have the same scale and dated at 0 ms. On the left side of maps, in blue, the activity of controlateral primary motor cortex involved in the response and on the right side of the map, in red, the activity of ipsilateral primary motor cortex involved in the response According to Grillner andhis colleagues (2005, 2013), the basal ganglia are strongly involved in the selection of basic motor programs (e.g., locomotion, chewing, swallowing, eye movements…), through a basic organization that has been conserved throughout vertebrate phylogeny, from lamprey to primates. The model of [START_REF] Keeler | Functional implications of dopamine D1 vs. D2 receptors: a 'prepare and select' model of the striatal direct vs. indirect pathways[END_REF], the data of [START_REF] Yoon | Delay period activity of the substantia nigra during proactive control of response selection as determined by a novel fMRI localization method[END_REF], and the present results suggest that the implication of basal ganglia in action selection might also concern more flexible experience-dependent motor programs.

A final comment is in order. Although the Laplacian transformation largely increases the spatial resolution of EEG data, it is not possible to spatially separate the subregions of SMAs, i.e., the pre-SMA and SMA proper [START_REF] Luppino | Multiple representations of body movements in mesial area 6 and the adjacent cingulate cortex: an intracortical microstimulation study in the macaque monkey[END_REF][START_REF] Matsuzaka | A motor area rostral to the supplementary motor area (presupplementary motor area) in the monkey: neuronal activity during a learned motor task[END_REF]Picard andStrick 1996, 2001). [START_REF] Larson | The effects of acute dopamine precursor depletion on the cognitive control functions of performance monitoring and conflict processing: an event-related potential (ERP) study[END_REF] reported that APTD does not affect the Error Negativity (note that we did not evidence either any effect of APTD on the Error Negativity in the present experiment [data not shown]). Now, it has been demonstrated with intracerebral electroencephalography in human subjects that the Error Negativity is primarily generated in SMA proper but not in pre-SMA [START_REF] Bonini | Action monitoring and medial frontal cortex: leading role of supplementary motor area[END_REF]. This suggests that SMA proper activity is not noticeably impaired by ATPD. As a consequence, it seems likely that the N-40 is generated in the pre-SMA. Although both areas receive disynaptic inputs from the basal ganglia via the thalamus, a differential sensitivity of SMA proper and pre-SMA to dopamine depletion is not necessarily surprising if one considers that pre-SMA and SMA proper are targeted by neurons located in neurochemically and spatially distinct regions of the internal segment of the globus pallidus [START_REF] Akkal | Supplementary motor area and presupplementary motor area: targets of basal ganglia and cerebellar output[END_REF]), a major output structure of the basal ganglia.

Two limitations of the present study must be noticed. First, considering that the model of [START_REF] Keeler | Functional implications of dopamine D1 vs. D2 receptors: a 'prepare and select' model of the striatal direct vs. indirect pathways[END_REF] not only assumes that the dopaminergic system is involved in response selection but also supposes that response selection of the appropriate response is achieved via the D2 system, the present study is unable to determine whether the effects of dopamine depletion are due to D1, D2 subreceptor types, or both; the same remark would also hold for [START_REF] Yoon | Delay period activity of the substantia nigra during proactive control of response selection as determined by a novel fMRI localization method[END_REF] results. Secondly, our ATPD manipulation is an all or none one, with no possibility to evidence Bdose-dependent effects.^Future pharmacological manipulations would allow dose-dependent manipulations with possibly no behavioral effects at lower doses and behavioral impairments at higher doses. Such manipulations would also allow separating at least in part D2 from D1 effects to test further the model of [START_REF] Keeler | Functional implications of dopamine D1 vs. D2 receptors: a 'prepare and select' model of the striatal direct vs. indirect pathways[END_REF].

To conclude, our results extend those of Yoon, who showed in a fMRI study that the dopaminergic system was finely sensitive to the complexity/simplicity of response selection during preparation. Thanks to the N-40, in accordance with the model of [START_REF] Keeler | Functional implications of dopamine D1 vs. D2 receptors: a 'prepare and select' model of the striatal direct vs. indirect pathways[END_REF], the present results directly indicate that the dopaminergic system is selectively involved in response selection per se, with little or no effect on response execution processes or proactive control of errors.

Fig. 1

 1 Fig. 1 Schematic representation of the task used by Yoon et al. (2015) and the present task. Congruency in the Yoon et al. (2015) task was between the arrow direction and the response side and in the present task between stimulus position and response side

Table 1

 1

	Amino acid plasmatic
	concentrations of phenylalanine	Amino acid	Before the absorption	After the absorption
			of the mixture	of the mixture
		Phenylalanine (μmol/l) placebo	60.3 ± 3.2	106.8 ± 6.1*
		Phenylalanine (μmol/l) depleted	59.8 ± 3.5	17.4 ± 3.6*
		Tyrosine (μmol/l) placebo	74.4 ± 4.1	272.7 ± 10.8*
		Tyrosine (μmol/l) depleted	71.7 ± 4.4	20.5 ± 4.4*

Laboratoire de Neurosciences Cognitives, Aix-Marseille Univ/CNRS, Marseille, France

Montreal Neurological Institute and Hospital, McGill University, Montreal, Quebec, Canada

Institut de Médecine Navale du Service de Santé des Armées, Toulon, France

Acknowledgements We thank Dominique Reybaud, Bruno Schmid, and the pharmacy personnel of the hospital Sainte Anne for their helpful technical contribution.

Funding information

The authors also gratefully acknowledge the financial support from the Institut de Recherches Biomédicales des Armées, France.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


Stephane Greff
email: stephane.greff@imbe.fr

Mayalen Zubia
email: mayalen.zubia@upf.pf

Claude Payri
email: claude.payri@ird.fr

Olivier P Thomas
email: olivier.thomas@nuigalway.ie

Thierry Perez
email: thierry.perez@imbe.fr

Stéphane Greff

Stéphane Greff

Chemogeography of the red macroalgae Asparagopsis: metabolomics, bioactivity, and relation to invasiveness

Keywords: Asparagopsis taxiformis, Macroalgal proliferations, Metabolomics, Transoceanic comparisons, Microtox®, UHPLC-HRMS

Introduction

Ecologists generally assume that biotic interactions are prominent under tropics [START_REF] Schemske | Is there a latitudinal gradient in the importance of biotic interactions?[END_REF] where species richness and biomass are considered higher [START_REF] Brown | Why are there so many species in the tropics?[END_REF][START_REF] Mannion | The latitudinal biodiversity gradient through deep time[END_REF]. The latitudinal gradient hypothesis (LGH) states that tropical plants inherit more defensive traits from higher pressures of competition, herbivory and parasitism than their temperate counterparts [START_REF] Coley | Comparison of herbivory and plant defenses in temperate and tropical broad-leaved forests[END_REF][START_REF] Coley | Herbivory and plant defenses in tropical forests[END_REF][START_REF] Schemske | Is there a latitudinal gradient in the importance of biotic interactions?[END_REF]). The same trend exists in marine ecosystems as temperate macroalgae are consumed overall twice more than the better defended tropical ones [START_REF] Bolser | Are tropical plants better defended? Palatability and defenses of temperate vs. tropical seaweeds[END_REF]. However, the assumption that both biotic interactions and defense metabolism are strongly related to latitudinal gradient, and resulting from co-evolutionary processes, still requires additional evidences [START_REF] Moles | Dogmatic is problematic: Interpreting evidence for latitudinal gradients in herbivory and defense[END_REF][START_REF] Moles | Is the notion that species interactions are stronger and more specialized in the tropics a zombie idea?[END_REF]. Studies did not show any relationship between herbivory pressure and latitudes [START_REF] Adams | A test of the latitudinal defense hypothesis: Herbivory, tannins and total phenolics in four North American tree species[END_REF][START_REF] Andrew | Herbivore damage along a latitudinal gradient: relative impacts of different feeding guilds[END_REF], and an opposite trend has also been demonstrated in some cases [START_REF] Del-Val | Seedling mortality and herbivory damage in subtropical and temperate populations: Testing the hypothesis of higher herbivore pressure toward the tropics[END_REF]. For instance, phenolic compounds in terrestrial [START_REF] Adams | A test of the latitudinal defense hypothesis: Herbivory, tannins and total phenolics in four North American tree species[END_REF]) and marine ecosystems seem to be equally present under low and high latitudes [START_REF] Targett | Biogeographic comparisons of marine algal polyphenolics: evidence against a latitudinal trend[END_REF][START_REF] Van Alstyne | The biogeography of polyphenolic compounds in marine macroalgae: Temperate brown algal defenses deter feeding by tropical herbivorous fishes[END_REF].

Chemical traits may also be related to environmental and/or biotic interactions changes [START_REF] Nylund | Metabolomic assessment of induced and activated chemical defence in the invasive red alga Gracilaria vermiculophylla[END_REF]. Several ecosystems are affected by introduction of nonindigenous species (NIS) which may disrupt biotic interactions [START_REF] Schaffelke | Impacts of introduced seaweeds[END_REF][START_REF] Simberloff | Impacts of biological invasions: What's what and the way forward[END_REF]. After the loss of specific competitors, NIS may reallocate the energy originally dedicated to defenses (specialized metabolism) into reproduction and growth (primary metabolism), and succeed in colonized environments [START_REF] Keane | Exotic plant invasions and the enemy release hypothesis[END_REF]. Interactions between NIS and native species may also modify chemical traits as it is argued by the novel weapon hypothesis (NWH) [START_REF] Callaway | Novel weapons: invasive success and the evolution of increased competitive ability[END_REF]. In addition, the production of defensive compounds may also be influenced by several abiotic factors such as temperature [START_REF] Ivanisevic | Biochemical trade-offs: evidence for ecologically linked secondary metabolism of the sponge Oscarella balibaloi[END_REF][START_REF] Reverter | Secondary metabolome variability and inducible chemical defenses in the Mediterranean sponge Aplysina cavernicola[END_REF], light [START_REF] Cronin | Effects of light and nutrient availability on the growth, secondary chemistry, and resistance to herbivory of two brown seaweeds[END_REF][START_REF] Deneb | Chemical defenses of marine organisms against solar radiation exposure Marine Chemical Ecology[END_REF][START_REF] Paul | The ecology of chemical defence in a filamentous marine red alga[END_REF], and nutrient availability [START_REF] Cronin | Effects of light and nutrient availability on the growth, secondary chemistry, and resistance to herbivory of two brown seaweeds[END_REF]. Moreover, internal factors such as reproductive stage (Ivanisevic et al. 2011a;[START_REF] Vergés | Sex and life-history stage alter herbivore responses to a chemically defended red alga[END_REF], ontogeny [START_REF] Paul | Simple growth patterns can create complex trajectories for the ontogeny of constitutive chemical defences in seaweeds[END_REF]) are globally subjected to seasonal variation and consequently they may affect the s pecialized metabolism ( Ivanisevic et al. 2011a).

The genus Asparagopsis (Rhodophyta, Bonnemaisoniaceae) is currently represented by two species, A. taxiformis (Delile) Trévisan de Saint-Léon and A. armata (Harvey) [START_REF] Andreakis | Asparagopsis taxiformis and Asparagopsis armata (Bonnemaisoniales, Rhodophyta): Genetic and morphological identification of Mediterranean populations[END_REF][START_REF] Dijoux | The more we search, the more we find: discovery of a new lineage and a new species complex in the genus Asparagopsis[END_REF]. Asparagopsis taxiformis is widespread in temperate, subtropical and tropical areas and, so far, six cryptic lineages with distinct geographic distributions have been described for this species [START_REF] Andreakis | Phylogeography of the invasive seaweed Asparagopsis (Bonnemaisoniales, Rhodophyta) reveals cryptic diversity[END_REF][START_REF] Andreakis | Endemic or introduced? Phylogeography of Asparagopsis (Florideophyceae) in Australia reveals multiple introductions and a new mitochondrial lineage[END_REF][START_REF] Dijoux | The more we search, the more we find: discovery of a new lineage and a new species complex in the genus Asparagopsis[END_REF]). Among them, the worldwide fragmented distribution pattern of A. taxiformis lineage two is explained by multiple introduction events, and in some places of the Southwestern Mediterranean Sea for instance, it is clearly invasive and outcompeting indigenous benthic organisms [START_REF] Altamirano | The invasive species Asparagopsis taxiformis (Bonnemaisoniales, Rhodophyta) on Andalusian coasts (Southern Spain): Reproductive stages, new records and invaded communities[END_REF][START_REF] Zanolla | The seasonal cycle of Asparagopsis taxiformis (Rhodophyta, Bonnemeaisoniaceae): key aspects of the ecology and physiology of the most invasive macroalga in Southern Spain[END_REF].

The genus Asparagopsis is known to biosynthesize about one hundred of halogenated volatile hydrocarbons containing one to four carbons with antimicrobial, antifeedant and cytotoxic properties [START_REF] Genovese | In vitro evaluation of antibacterial activity of Asparagopsis taxiformis from the Straits of Messina against pathogens relevant in aquaculture[END_REF][START_REF] Kladi | Volatile halogenated metabolites from marine red algae[END_REF]Paul et al. 2006b). Assessment of resources allocated to defense traits can be obtained through analysis of the specialized metabolism using metabolomics. Another way to evaluate resources allocated to defense traits is to measure the bioactivity of organismal extract as a proxy of defense-related compounds biosynthesis. The Microtox® assay is a simple, efficient and rapid method that highly correlates with other biological tests [START_REF] Botsford | A comparison of ecotoxicological tests[END_REF]. Trade-off between the specialized metabolism, and the primary metabolism dedicated to essential biochemical processes such as growth and reproduction can be assessed by this approach [START_REF] Ivanisevic | Biochemical trade-offs: evidence for ecologically linked secondary metabolism of the sponge Oscarella balibaloi[END_REF]). Bioactivities of extracts can be directly correlated to the expression level of targeted metabolites [START_REF] Cachet | Metabolomic profiling reveals deep chemical divergence between two morphotypes of the zoanthid Parazoanthus axinellae[END_REF][START_REF] Martí | Quantitative assessment of natural toxicity in sponges: toxicity bioassay versus compound quantification[END_REF][START_REF] Reverter | Secondary metabolome variability and inducible chemical defenses in the Mediterranean sponge Aplysina cavernicola[END_REF], and metabotypes were shown to explain bioactivity patterns [START_REF] Ivanisevic | Biochemical trade-offs: evidence for ecologically linked secondary metabolism of the sponge Oscarella balibaloi[END_REF]). However, metabolomics doesn't match necessarily bioactivity assessment. Indeed, metabolomics provides an overall picture of chemical complexity of a biological matrix, this picture being dependent of the selected technique (MS or NMR), but in any case without any indication of putative synergetic or antagonistic effects of the detected compounds. On the other hand, an assay such as the Microtox® integrates all putative synergetic or antagonistic effects of the extracted compounds, but the obtained value is only a proxy depending on the specificity of the model bacterial strain response.

The first objective of our study was to assess macroalgal investment in defensive traits using two non-equivalent approaches, UHPLC-HRMS metabolic fingerprinting and biogeographic variations of macroalgal bioactivities assessed with the Microtox® assay. The second objective was to understand how environmental factors (temperature, light) may influence macroalgal defensive traits. Finally, we also have evaluated the relationship between bioactivities and the status of the macroalga, its origin (introduced vs. native) and its cover together as an indicator of invasiveness, in order to relate the involvement of macroalgal chemical defenses in its proliferation trait.

Methods

Biological Material

Among the six different lineages of A. taxiformis (Delile) Trevisan de Saint-Léon (Rhodophyta, Bonnemaisoniaceae) [START_REF] Andreakis | Phylogeography of the invasive seaweed Asparagopsis (Bonnemaisoniales, Rhodophyta) reveals cryptic diversity[END_REF][START_REF] Andreakis | Endemic or introduced? Phylogeography of Asparagopsis (Florideophyceae) in Australia reveals multiple introductions and a new mitochondrial lineage[END_REF][START_REF] Dijoux | The more we search, the more we find: discovery of a new lineage and a new species complex in the genus Asparagopsis[END_REF], only five were considered in this study. This alga can cover hard and soft substrate from 0 to 45 m depth both in temperate and tropical waters. Asparagopsis armata (Harvey), a species distributed worldwide and currently composed of two distinct genetic clades [START_REF] Dijoux | The more we search, the more we find: discovery of a new lineage and a new species complex in the genus Asparagopsis[END_REF], was also considered in this study. Only one lineage mostly growing on hard substrates at shallow depth was investigated. The genus is dioecious. Gametophytes stages present distinct male and female individuals, which alternates with a heteromorphic tetrasporophyte "Falkenbergia stage". In this study, we focused on the gametophyte stage of the macroalgae.

Sampling

A total of 289 individuals of A. taxiformis gametophytic stage were collected in 21 stations selected in 10 sites from two zones (temperate and tropical), from October 2012 to April 2015 (Table 1). Sampled stations presented highly variable A. taxiformis covers. Three classes of Asparagopsis cover were determined by visual assessment: low, 0-35%, medium, 35-65%, high 65-100%. Asparagopsis armata was sampled in South of Spain where it lives in sympatry with A. taxiformis. Two temporal samplings of A. taxiformis were performed in Réunion (Saint Leu, four dates from October 2012 to July 2013) and in France (La Ciotat, six dates from November 2013 to April 2015).

Metabolite extraction

After collection, samples were transported in a cooler, stored at -20 °C before freeze-drying. Dried samples were preserved in silica gel and sent to Marseille (France). Each sample was then individually ground into a fine powder using a blender (Retsch® MM400, 30 Hz during 30 s). One hundred milligram of each sample were extracted 3 times with 2 mL of MeOH/CH 2 Cl 2 1:1 (v/v) in an ultrasonic bath (1 min) at room temperature. The filtrates (PTFE, 0.22 µm, Restek®) were pooled and concentrated to dryness, adsorbing extracts on C18 silica particles (100 mg, non-end-capped C18 Polygoprep 60-50, Macherey-Nagel®). The extracts were then subjected to SPE (Strata C18-E, 500 mg, 6 mL, Phenomenex®) eluting with H 2 O, MeOH, and CH 2 Cl 2 (5 mL of each) after cartridge cleaning (10 mL MeOH) and conditioning (10 mL H 2 O). MeOH fractions were evaporated to dryness, resuspended in 2 mL of MeOH prior to metabolomic analyses by UHPLC-QqTOF. After this first analysis, the same macroalgal extracts were concentrated to dryness ready to be used for bioactivity assessment using the Microtox® assay.

Metabolomic analyses

Chemicals

Methanol, dichloromethane and acetonitrile of analytical quality were purchased from Sigma-Aldrich (Chroma-solv®, gradient grade). Formic acid and ammonium formate (LC-MS additives, Ultra grade) were provided by Fluka.

LC-MS analyses

Analyses were performed on an UHPLC-QqToF instrument: UHPLC is equipped with RS Pump, autosampler and thermostated column compartment and UV diode array (Dionex Ultimate 3000, Thermo Scientific®) and coupled to a high resolution mass spectrometer (MS) equipped with an ESI source (Impact II, Bruker Daltonics®). Mass spectra were acquired in positive and negative mode consecutively. Elution rate was set to 0.5 mL min -1 at a constant temperature of 40 °C. Injection volume was set to 10 µL. Chromatographic solvents were composed of A: water with 0.1% formic acid (positive mode), or 10 mM ammonium formate (negative mode), and B: acetonitrile/water (95:5) with the same respective additives. UHPLC separation occurs on an Acclaim RSLC C18 column (2.1 × 100 mm, 2.2 µm, Thermo Scientific®). According to the study of spatial and temporal patterns, chromatographic elution gradients were adjusted to improve peak resolution using a pooled sample. Two chromatographic elution gradients were thus applied. For the study of spatial patterns, the program was set up at 40% B during 2 min, followed by a linear gradient up to 100% B during 8 min, then maintained 4 min in isocratic mode. The analysis was followed by a return to initial conditions for column equilibration during 3 min for a total runtime of 17 min. For the study of temporal patterns, the program was set up at 2% B during 1 min, followed by a linear gradient up to 80% B during 5 min, then maintained 6 min in isocratic mode at 80% B. The analysis was followed by a phase of 100% B during 4 min and equilibration during 4 min for a total runtime of 20 min. Analyses were processed in separate batches for the study of spatial and temporal variation of the metabotypes. Macroalgal extracts were randomly injected according to sampling sites or dates, alternating the pooled sample injected every 6 samples to realize inter and intra-batch calibration due to MS shift over time. MS parameters were set as follows for positive mode (and negative mode): nebulizer gas, N 2 at 31 psi (51 psi), dry gas N 2 at 8 L min -1 (12 L min -1 ), capillary temperature at 200 °C and voltage at 2500 V (3000 V). Data were acquired at 2 Hz in full scan mode from 50 to 1200 amu. Mass spectrometer was systematically calibrated with formate/acetate solution forming clusters on the studied mass range before a full set of analysis. The same calibration solution was automatically injected before each sample for internal mass calibration. Data-dependent acquisition MS 2 experiments were also conducted (renewed every three major precursors) on some samples of each location.

Data analyses

Constructor raw analyses were automatically calibrated using internal calibrant, before exporting data in netCDF files (centroid mode) using Bruker Compass DataAnalysis 4.3. All converted analysis were then processed by XCMS software [START_REF] Smith | XCMS: processing mass spectrometry data for metabolite profiling using nonlinear peak alignment, matching, and identification[END_REF]) under R (R_Core_Team 2013), using the different steps necessary to generate the final data matrix: (1) Peak picking (peakwidth = c [START_REF] Cardigos | Non-indigenous marine species of the Azores[END_REF]20), ppm = 2) without threshold prefilter [START_REF] Patti | Meta-analysis of untargeted metabolomic data from multiple profiling experiments[END_REF]), ( 2) retention time correction (method = "obiwarp"), (3) grouping (bw = 10, minfrac = 0.3, minsamp = 1), (4) Fillpeaks, and finally (5) report and data matrix generation transferred to Excel. Each individual ion was finally normalized (if necessary according to the drift of equivalent ion of pooled samples) and injection order (van der [START_REF] Van Der Kloet | Analytical error reduction using single point calibration for accurate and precise metabolomic phenotyping[END_REF]. Data were calibrated between batches (inter-batch calibration for the study of spatial patterns) by dividing each ion by the intra-batch mean value of pooled-sample ions, and multiplying by the total mean value for all batches [START_REF] Ejigu | Evaluation of normalization methods to pave the way towards large-scale LC-MS-based metabolomics profiling experiments[END_REF]. Metabolites were annotated with constructor software (Bruker Compass DataAnalysis 4.3).

Bioactivity assays

Bioactivities of macroalgal extracts were assessed using the standardized Microtox® assay (Microbics, USA). This ecotoxicological method measures the effect of compounds on the respiratory metabolism of Allivibrio fischeri which is correlated to the intensity of its natural bioluminescence [START_REF] Johnson | Microtox® acute toxicity test[END_REF]. Extracts were initially prepared at 2 mg mL -1 in artificial seawater with 2% of acetone to facilitate dissolution, and then diluted (twofold) three times in order to test their effect on bacteria and to draw EC 50 curves. EC 50 expressed in µg mL -1 represents the a According to the definition of an "introduction" by [START_REF] Boudouresque | Les espèces introduites et invasives en milieu marin, 3 edn[END_REF]: transportation favored directly or indirectly by humans, biogeographical discontinuity with native range and established (self-sustaining population). b Cover: low (up to 35%), medium (from 35 to 65%), high (over 65%). In summary, high probability of introduction together with high cover is considered equivalent to high invasiveness concentration that decreases half of the initial luminescence after 5 min of exposure to extracts.

Environmental factors

Sea surface temperature (SST in °C) and Photosynthetically available radiation (PAR in mol m -2 day -1 ) were obtained from NASA GES DISC for all sites (http://giovanni.gsfc.nasa.gov). In France, supplementary abiotic factors related to water chemistry such as ammonium (NH 4 + ), nitrate (NO 3 -), and phosphate (PO 4 3-) concentrations (in µmol L -1 ) were provided by SOMLIT (http://somlit. epoc.u-bordeaux1.fr/fr/).

Statistical analyses

Principal components analysis (PCA) were realized using the "ade4 package" [START_REF] Dray | The ade4 package: implementing the duality diagram for ecologists[END_REF]. PCA were centered (the mean ion intensity of all samples was subtracted to each sample ion intensity) and normalized (consequently divided by the relative standard deviation of ion intensity of all samples). PERMANOVA (adonis function, 1e 5 permutations) and ANalysis OF SIMilarity (ANO-SIM function using Euclidean distances) were performed with the "vegan package" [START_REF] Oksanen | vegan: community ecology package. R package version 2[END_REF]. PLS-DA were realized using the "RVAideMemoire package" [START_REF] Hervé | RVAideMemoire: Diverse Basic Statistical and Graphical Functions[END_REF] on scaled raw data according to zones and unscaled log-transformed data according to sites. Permutational test based on cross model validation procedures (MVA.test and pairwise.MVA.test) were used to test differences between groups: outer loop fivefold cross-validation, inner loop fourfold cross-validation according to zones and sites [START_REF] Szymanska | Double-check: validation of diagnostic statistics for PLS-DA models in metabolomics studies[END_REF]. Very important peaks (VIPs) were determined according to the PLS-DA loading plots. Non-parametric analysis (Kruskal-Wallis test followed by Steel-Dwass-Critchlow-Fligner post-hoc test, and Mann-Whitney) were performed on XLSTAT version 2015.4.01.20575 to test differences of macroalgal bioactivities according to zones and sites. The relationships of macroalgal bioactivities against A. taxiformis cover, latitudes or environmental factors were assessed using Spearman's correlations rank test (Rs) using XLSTAT.

Results

Spatial variation of the macroalgal chemical profiles and bioactivities

All the macroalgal metabotypes were plotted on principal component analysis (PCA) taking into account 2683 negative and 2182 positive ions. The PCA shows that the global inertia remains low with 11.9% of explanation of the variability (Fig. 1). Variances on axis 1-3 and 2-3 of the PCA show similar values with 11.2% and 9.3%, respectively. But the divergence between groups (zones and sites) is more evident along the 2-3 axis. Whereas the difference between macroalgal metabotypes in tropical and temperate zones is not statistically supported (PERMANOVA, F = 2.7, p = 0.064), a significant difference between sites is recorded (PERMANOVA, F = 2.9, p = 0.003), with a weak Pearson correlation factor (R 2 = 0.13). A similarity test between sites confirmed that macroalgae sampled in Azores and France are significantly different from all other sites, as well as for macroalgae sampled in Martinique and New Caledonia (ANOSIM, R = 0.281, p < 0.001) (Fig S1

and Table S1 in Supporting Information). Eight metabolite features were selected as chemomarkers with regards to the congruence of ions detected in both negative and positive modes from the PPLS-DA loading plots. They partly explain the dispersion of groups that differentiate temperate metabotypes from tropical ones (PPLS-DA, NMC = 2.5%, p = 0.001) (Fig S2). Differentiation of groups was also effective for metabotypes of algae sampled from the different sites (PPLS-DA, NMC = 31.8%, p = 0.001) (Fig S3

and Table S2). Most probable raw formula of biomarkers did not match with any known compounds from the genus Asparagopsis (Table S3).

Macroalgal bioactivities are negatively correlated with latitudes (Rs = -0.148, R 2 = 0.02, p = 0.041). Overall A. taxiformis from temperate zones show higher bioactivities (EC 50 = 32 ± 3 µg mL -1 , mean ± SE) than those from tropical zones (85 ± 5 µg mL -1 , Mann-Whitney, U = 4256, p < 0.001, Figs. 2,3a). Macroalgal bioactivities also differ significantly according to sampling sites (Kruskal-Wallis, K = 90, p < 0.001, Fig. 2b). EC 50 ranged from 20 ± 4 µg mL -1 (Azores) to 117 ± 15 µg mL -1 (French Polynesia). Similarly to macroalgal bioactivities found in Azores, high values were recorded in France (31 ± 4 µg mL -1 ), Algeria (33 ± 5 µg mL -1 ) and Spain (52 ± 13 µg mL -1 ). In comparison, A. armata sampled in South of Spain did not show significant different EC 50 values from A. taxiformis sampled in France, Algeria and Spain (ESP arm , 24 ± 4 µg mL -1 , Steel-Dwass-Critchlow-Fligner post hoc test, p > 0.05).

Macroalgae from Martinique (114 ± 24 µg mL -1 ) and French Polynesia (117 ± 15 µg mL -1 ) showed the lowest bioactivities, whereas macroalgae from Mayotte showed the highest values among the tropical macroalgae (57 ± 8 µg mL -1 ). Asparagopsis taxiformis from other tropical sites (New Caledonia, Réunion and Guadeloupe) exhibited intermediate bioactivities (respectively 72 ± 7 µg mL -1 , 80 ± 10 µg mL -1 , 80 ± 22 µg mL -1 ).

No relationship between the spatial pattern of variability in the macroalgal bioactivity and the macroalgal cover has been established (Rs = 0.061, R 2 = 0.004, p = 0.336). This spatial pattern in macroalgal bioactivities is actually negatively correlated with SST (Rs = -0.428, R 2 = 0.18, p < 0.001) and PAR (Rs = -0.37, R 2 = 0.14, p < 0.001) which explain respectively 18 and 14% of the overall variability (Fig. 1; Table 2).

Temporal variation of the macroalgal chemical profiles and bioactivities

Macroalgae from France and Réunion displayed distinct metabotypes that varied in time, with overall a much higher variability recorded in Réunion and no clear pattern of seasonal variation in France (Fig. 3a,b). PCAs show that inertia are globally higher when assessing the spatial variability, with about 19% explained in France and 24% in Réunion. Although we were able to distinguish several metabotypes in these time series, no clear chemomarkers were identified to explain this variability. This divergence likely relies on several minor ions.

The EC 50 values for A. taxiformis from France range from 13 ± 3 to 37 ± 5 µg mL -1 (mean ± SE) revealing a high bioactivity all along the year (Fig. 4a), individuals sampled in January 2015 exhibiting the lowest bioactivity values recorded for this site (EC 50 = 37 ± 5 µg mL -1 ). This temporal pattern of variability is positively correlated to SST variations (Rs = 0.287, R 2 = 0.08, p = 0.02) (PAR, p > 0.05), and negatively correlated to variation in ammonium and nitrate concentrations (Rs = -0.373, R 2 = 0.14, p = 0.003 for NH 4 + ; Rs = -0.435, R 2 = 0.19, p < 0.001 for NO 3 -; Table 2). In Réunion, macroalgal bioactivities show a higher variability than those recorded for temperate site (Kruskal-Wallis, K = 29, p < 0.001; Fig. 4b). The lowest values (EC 50 = 204 ± 13 µg mL -1 ) were recorded in January when the seawater temperature is the highest (monthly S4 for details according to sites) mean SST of 27.1 °C, Table S5), whereas the highest values (EC 50 = 14 ± 3 µg mL -1 ) were recorded in July when the seawater temperature is lower (monthly mean SST of 23.5 °C). There is thus a negative correlation between the macroalgal bioactivity, the SST and the PAR variability (Rs = -0.729, R 2 = 0.53, p < 0.001 and Rs = -0.532, R 2 = 0.28, p < 0.001, for SST and PAR respectively).

Discussion

Applying LC-MS-based metabolomics to halogenated metabolites

The genus Asparagopsis is known to biosynthesize about one hundred of halogenated volatile organic compounds [START_REF] Kladi | Volatile halogenated metabolites from marine red algae[END_REF]). Whereas the major metabolites are assumed to be low molecular weight brominated compounds (Paul et al. 2006a), the metabolomic approach used in this study mostly detected non-halogenated metabolites. This might be explained by the volatility of these small compounds that are mainly detected using GC-MS analysis. Higher molecular weight metabolites with six carbons named mahorones were reported from this species [START_REF] Greff | Mahorones, highly brominated cyclopentenones from the red alga Asparagopsis taxiformis[END_REF]. Targeted search of the mahorones in collected gametophytes revealed the presence of 5-bromomahorone in almost all samples without any clear pattern of distribution between samples. The second mahorone was not detected maybe because of difficulties in the ionization process of these molecules as described by [START_REF] Greff | Mahorones, highly brominated cyclopentenones from the red alga Asparagopsis taxiformis[END_REF].

In this study, brominated and iodinated metabolites, were only evidenced by the release of bromide and iodide in the negative mode. Electrospray ionization is strongly dependent of metabolite physical and chemical properties. In negative mode, the detection of halogenated metabolites are not favored as the electrons may be trapped by halogens, rendering halogenated metabolites unstable and undetectable to mass spectrometer (except for halide ions). A metabolomic approach using HRMS is thus suitable for the detection of easily ionizable metabolites present in the macroalgae, but there is a limitation when the major specialized metabolites are highly halogenated.

Relationship between metabotypes and bioactivities

Although, various metabotypes were clearly discriminated, the divergence is due to a high number of minor ions. In this study, phenotypes of temperate macroalgae, especially A. armata sampled in Spain and A. taxiformis sampled in Spain and Algeria, distinguish mostly by the presence of some metabolite features named MF1-MF8 not previously described for these species. Macroalgae sampled in temperate environments evidenced higher bioactivities than those sampled under tropical environments indicating that macroalgal investment in defense was greater under higher latitudes. So far, temperate A. taxiformis (France, Azores, Spain and Algeria) is mainly represented by the introduced lineage 2 (L2) [START_REF] Andreakis | Phylogeography of the invasive seaweed Asparagopsis (Bonnemaisoniales, Rhodophyta) reveals cryptic diversity[END_REF][START_REF] Dijoux | The more we search, the more we find: discovery of a new lineage and a new species complex in the genus Asparagopsis[END_REF] suggesting that this NIS can modify its investment in chemical traits. Both species, A. taxiformis and A. armata, sampled in South Spain showed closer phenotypes and bioactivities than A. taxiformis sampled at a larger geographic scale. This outcome suggests that macroalgal phenotypes are more driven by environmental factors, at least partly related to microbial communities, than to genetic factors. This phenotypic variability related to the exposome was already known at the morphological level, as a given genetic lineage or population can include various morphotypes [START_REF] Dijoux | La diversité des algues rouges du genre Asparagopsis en Nouvelle-Calédonie : approches in situ et moléculaires[END_REF][START_REF] Monro | The evolvability of growth form in a clonal seaweed[END_REF], and the morphotype variability could never be explained by genetics [START_REF] Dijoux | La diversité des algues rouges du genre Asparagopsis en Nouvelle-Calédonie : approches in situ et moléculaires[END_REF]. Although MS metabolomics was applied with success to a rather good number of chemotaxonomy or chemosystematics works, this study did not allow to discriminate macroalgal lineages. The unusual ionization processes of the major and highly halogenated specialized metabolites produced by these species might be one of the main explanation, calling thus to use other technical approaches. Besides metabolomics, bioactivity assessment using the Microtox® assay appeared as a relevant complement to our MS approach in order to detect putative shift in macroalgal chemical diversity and its related bioactivity.

In addition, the results of the Microtox® analyses, used as a proxy of the production of chemical defenses, are not in accordance with the Latitudinal Gradient Hypothesis (LGH) used on land where plants allocate more in defensive traits under lower latitudes. It also shows that environmental factors are driving-forces that can strongly influence the specialized metabolism and its related bioactivity or putative ecological function [START_REF] Pelletreau | New perspectives for addressing patterns of secondary metabolites in marine macroalgae[END_REF][START_REF] Puglisi | Marine chemical ecology in benthic environments[END_REF][START_REF] Putz | Chemical defence in marine ecosystems[END_REF]. A higher herbivory pressure in tropical ecosystems than in temperate ones can rely on the species richness and biomass of tropical ecosystems [START_REF] Brown | Why are there so many species in the tropics?[END_REF][START_REF] González-Bergonzoni | Meta-analysis shows a consistent and strong latitudinal pattern in fish omnivory across ecosystems[END_REF], but also to a stronger resistance of herbivores to plant metabolites [START_REF] Craft | Biogeographic and phylogenetic effects on feeding resistance of generalist herbivores toward plant chemical defenses[END_REF]. Previous study conducted with A. armata demonstrated that an increase in toxicity towards bacteria was related to the amount of bioactive halogenated compounds (Paul et al. 2006a). The halogenation process was also shown determinant for the deterrence of non-specialized herbivores (Paul et al. 2006b;[START_REF] Rogers | Ecology of the sea hare Aplysia parvula (Opisthobranchia) in New South Wales, Australia[END_REF][START_REF] Vergés | Sex and life-history stage alter herbivore responses to a chemically defended red alga[END_REF]) and the abalone Haliotis rubra (Paul et al. 2006b;Shepherd andSteinberg 1992 in Paul 2006) are known to graze A. armata, and only the sea hare Aplysia fasciata was reported to feed on A. taxiformis [START_REF] Altamirano | The invasive species Asparagopsis taxiformis (Bonnemaisoniales, Rhodophyta) on Andalusian coasts (Southern Spain): Reproductive stages, new records and invaded communities[END_REF].

Competition for space might also promote defensive traits as macroalgae can be abundant in temperate infralittoral zones [START_REF] Mineur | European seaweeds under pressure: Consequences for communities and ecosystem functioning[END_REF][START_REF] Vermeij | Biogeography and adaptation: Patterns of marine life[END_REF]. For A. taxiformis in the Mediterranean Sea, the pressure of competition might be expected to be rather high in spring when productivity is the highest [START_REF] Pinedo | Seasonal dynamics of upper sublittoral assemblages on Mediterranean rocky shores along a eutrophication gradient[END_REF], but our temporal survey showed rather similar bioactivities all along the year. If macroalgal-macroalgal interactions can induce defensive metabolites biosynthesis, it remains however difficult to explain why A. taxiformis maintains such a high level of defensive traits whereas these interactions are supposed to decrease. Competition is closely related to light availability. Under temperate latitudes, the photophilic community is generally more bioactive than the hemisciaphilic communities indicating that light plays a key role in bioactivity and the biosynthesis of defense related metabolites [START_REF] Martí | Seasonal and spatial variation of species toxicity in Mediterranean seaweed communities: correlation to biotic and abiotic factors[END_REF][START_REF] Mtolera | Stress-induced production of volatile halogenated organic compounds in Eucheuma denticulatum (Rhodophyta) caused by elevated pH and high light intensities[END_REF]. [START_REF] Paul | The ecology of chemical defence in a filamentous marine red alga[END_REF] demonstrated that the production of specialized metabolites was not costly for A. armata when the light is not limited, as biosynthesis was positively correlated to growth. Yet, light is scarcely limited except when competition with fleshy macroalgae reaches a maximum. Under tropics, high irradiance should lead to the synthesis of defense metabolites as revealed for the Rhodophyta Eucheuma denticulatum [START_REF] Mtolera | Stress-induced production of volatile halogenated organic compounds in Eucheuma denticulatum (Rhodophyta) caused by elevated pH and high light intensities[END_REF], but excessive irradiance may also stress macroalgae leading to biosynthesis switch [START_REF] Cronin | Effects of light and nutrient availability on the growth, secondary chemistry, and resistance to herbivory of two brown seaweeds[END_REF].

Taking all these factors together, seasonal bioactivity variation can give some clues. Surprisingly, the highest variation of macroalgal bioactivities was displayed by A. taxiformis in tropical region (Réunion) while seasonality was quite weak with a lower temperature range (5 °C) and high irradiance all along the year. During austral winter (with SST of 23-24 °C), A. taxiformis from Réunion showed bioactivities equivalent to temperate zones whereas the lowest bioactivities were displayed in austral summer when the water temperature was higher (26-27 °C). Asparagopsis taxiformis thermal tolerance was tested up to 30 °C (Padilla-Gamino and Carpenter 2007). However, high temperatures coupled to high irradiance and low nutrient levels that characterize tropical environments [START_REF] Vermeij | Biogeography and adaptation: Patterns of marine life[END_REF]) may lead to metabolic alterations as suggested by [START_REF] Cronin | Effects of light and nutrient availability on the growth, secondary chemistry, and resistance to herbivory of two brown seaweeds[END_REF]. Thus, a way to explain the higher defensive traits in temperate environments is to consider that maintaining a high level of defensive trait may not be so costly as long as light and nutrients are available, and temperature physiologically adequate.

Relationship between macroalgal bioactivities and invasiveness

In temperate regions, A. taxiformis was reported to be recently introduced in many places. In Azores, A. taxiformis spread all around the islands until the last 90′ and it is now well established [START_REF] Cardigos | Non-indigenous marine species of the Azores[END_REF][START_REF] Chainho | Non-indigenous species in Portuguese coastal areas, coastal lagoons, estuaries and islands[END_REF][START_REF] Micael | Tracking macroalgae introductions in North Atlantic oceanic islands[END_REF]). The last report on the worldwide distribution of A. taxiformis genetic lineages confirmed the presence of the introduced L2 in two Azorean Islands [START_REF] Dijoux | The more we search, the more we find: discovery of a new lineage and a new species complex in the genus Asparagopsis[END_REF]). In the Western Mediterranean Sea, only the L2 has been recorded so far [START_REF] Andreakis | Asparagopsis taxiformis and Asparagopsis armata (Bonnemaisoniales, Rhodophyta): Genetic and morphological identification of Mediterranean populations[END_REF][START_REF] Andreakis | Phylogeography of the invasive seaweed Asparagopsis (Bonnemaisoniales, Rhodophyta) reveals cryptic diversity[END_REF][START_REF] Andreakis | High genetic diversity and connectivity in the polyploid invasive seaweed Asparagopsis taxiformis (Bonnemaisoniales) in the Mediterranean, explored with microsatellite alleles and multilocus genotypes[END_REF][START_REF] Dijoux | The more we search, the more we find: discovery of a new lineage and a new species complex in the genus Asparagopsis[END_REF], but an invasive trait is not recorded everywhere [START_REF] Zenetos | Alien species in the Mediterranean Sea by 2012. A contribution to the application of European Union's Marine Strategy Framework Directive (MSFD). Part 2[END_REF]. In Alboran Sea, where this species has spread quickly since the late XXth century, A. taxiformis can form monospecific stands in several places along the Iberian coasts [START_REF] Altamirano | The invasive species Asparagopsis taxiformis (Bonnemaisoniales, Rhodophyta) on Andalusian coasts (Southern Spain): Reproductive stages, new records and invaded communities[END_REF][START_REF] Altamirano | New records for the benthic marine flora of Chafarinas Islands (Alboran Sea, Western Mediterranean)[END_REF]. In the same biogeographic region, a high cover of A. taxiformis was also recorded off the Algerian coast, while the macroalga is poorly distributed at Ceuta and the Strait of Gibraltar. Thus, for the widespread lineage 2 which is considered as invasive in some regions of the Mediterranean Sea and North Atlantic [START_REF] Altamirano | The invasive species Asparagopsis taxiformis (Bonnemaisoniales, Rhodophyta) on Andalusian coasts (Southern Spain): Reproductive stages, new records and invaded communities[END_REF][START_REF] Micael | Tracking macroalgae introductions in North Atlantic oceanic islands[END_REF][START_REF] Streftaris | Alien marine species in the Mediterranean-the 100 'Worst Invasives' and their impact[END_REF], we observed highly variable fate in the indigenous benthic community, proliferating in some places and rather discreet in others. This observation can be extended to different lineages present in other geographic context, which tends to indicate no link between macroalgal bioactivities, their metabotypes and their invasiveness. It is likely that other physiological traits, compared to indigenous sessile organisms, may explain its success in certain habitats, such as special efficiencies for up-taking nutrients, for spreading a specific life cycle stage, or for resisting to environmental stress thanks to the polyploid status of the thalli.

Fig. 1

 1 Fig. 1 Principal component analysis (PCA) of methanolic macroalgal extracts analyzed in UHPLC-QqToF (positive and negative modes) according to zones (temperate versus tropical) and sites PYF: French Polynesia, MTQ: Martinique, GUA: Guadeloupe, MYT: Mayotte, REU: Réunion, NCL: New Caledonia, AZO: Azores, FRA: France, DZA: Algeria; ESP: Spain with A. taxiformis, ESP tax and

Fig. 2

 2 Fig. 2 Mean bioactivities (±SE) of methanolic macroalgal extracts measured with Microtox® ecotoxicological assay according to (a) sampling zones: tropical vs. temperate and (b) sites: PYF: French Polynesia, NCL: New Caledonia, REU: Réunion, MYT: Mayotte, MTQ: Martinique, GUA: Guadeloupe, AZO: Azores, FRA, France, DZA: Algeria, ESP: Spain. Numbers of samples tested written in bars. Comparisons between zones were achieved with Mann-Whitney test. Comparisons between sites were achieved with Kruskal-Wallis test followed by Steel-Dwass-Critchlow-Fligner post hoc test. Letters figure differences between groups

Fig. 3

 3 Fig. 3 Principal component analysis (PCA) of methanolic macroalgal extracts analyzed in UHPLC-QqToF according to temporal variation for two sites/zones: (a) France for temperate zone and (b) Réunion for tropical zone. EC 50 (in µg mL -1 ) of A. taxiformis opposite to

Fig. 4

 4 Fig. 4 Mean bioactivities (± SE) of methanolic macroalgal extracts measured with Microtox® assay according to season at (a) La Ciotat (France) and (b) Saint Leu (Réunion). Numbers of samples tested are written in bars. Comparisons were achieved using Kruskal-Wallis test followed by Steel-Dwass-Critchlow-Fligner post hoc test. Letters figure differences between groups

Table 1

 1 Sampling sites of Asparagopsis spp. around the world for the study of spatial and temporal variation of macroalgal bioactivities and chemical phenotypes

	Cover b Sampling date Latitude Longitude Depth Sampling effort		A. taxiformis A. armata	high 07/11/2012 38°31.309′N 28°38.315′W -9	high 04/09/2013 36°47.587′N 3°21.078′E -12	high 25/04/2013 11 36°43.276′N 3°44.119′O 3-25 5	low 26/04/2013 35°53.848′N 5°18.479′O -1	low 27/04/2013 35°53.936′N 5°18.495′O -1	low 21/05/2013 43°9.957′N 5°36.539′E 5-8 9	low 14/04/2014 16°9.603′N 61°32.74′W 10-12 10	low 07/03/2014 14°28.86′N 61°5.095′W 24-24 7	high 06/04/2013 12°49.277'S 45°17.406′E -10	high 06/04/2013 12°59.987'S 45°6.543′E -9	low 29/10/2012 21°10.157'S 55°17.102′E <1 20	low 31/01/2013 22°18.888'S 166°26.085′E 0.5-2 2	medium 01/02/2013 22°20.816'S 166°13.954′E 6-14 11	medium 01/02/2013 22°20.836'S 166°13.906′E 10-35 5	low 4-5/02/2013 8 20°13.23'S 165°17.122′E 6-12	low 06/02/2013 3 20°40.403'S 164°15.431′E -	medium 07/02/2013 26 21°41.376'S 165°27.735′E 5-35	low 22/11/2012 6 17°43.681'S 149°35.399′W -	low 22/11/2012 6 17°32.974'S 149°37.852′W -	low 23/11/2012 5 17°32.179'S 149°35.703′W -	low 26/11/2012 10 17°36.661'S 149°37.296′W 4-35	low 1-8/02/2013 16 23° 10.051'S 134° 55.839′W 3-5		08/11/2013 8 43°9.957′N 5°36.539′E 5-8	08/01/2014 10 43°9.957′N 5°36.539′E 5-8	06/05/2014 7 43°9.957′N 5°36.539′E 5-8	01/07/2014 10 43°9.957′N 5°36.526′E 5-8	30/09/2014 10 43°9.957′N 5°36.526′E 5-8	01/08/2015 9 43°9.957′N 5°36.526′E 5-8	15/04/2015 5 43°9.957′N 5°36.526′E 5-8	04/10/2012 10 21°10.157'S 55°17.102′E <1	30/01/2013 10 21°10.157'S 55°17.102′E <1	25/04/2013 9 21°10.157'S 55°17.102′E <1	03/07/2013 21°10.157'S 55°17.102′E <1
	Probability of introduction a		high	high	high	high	high	high	uncertain	uncertain	uncertain	uncertain	uncertain	low	low	low	low	low	low	low	low	low	low	low
	Zone Sites Stations Most prob-	able lineage	Spatial variation	Temperate Azores -L2	Algeria Ilôt de Bounettah L2	Spain La Herradura L2	Ceuta Ciclon de Tierra L2	Ceuta Ciclon de Fuera L2	France La Ciotat L2	Tropical Guadeloupe Caye a Dupont L3	Martinique Anses d'Arlet L3	Mayotte Aéroport L4	Kani kéli L4	Réunion Saint Leu Ravine L4	New Caledonia Ilot Canard L5	Dumbéa L5	Dumbéa L5	Touho L5	Koumac Kendec L5	Bourail L5	French Polynesia Paea L4	Faaa1 L4	Faaa2 L4	Taapuna L4	Mangareva L4 (L5)	Temporal variation	Temperate France La Ciotat-Mugel L2	La Ciotat-Mugel L2	La Ciotat-Mugel L2	La Ciotat-Mugel L2	La Ciotat-Mugel L2	La Ciotat-Mugel L2	La Ciotat-Mugel L2	Tropical Réunion Saint Leu Ravine L4	Saint Leu Ravine L4	Saint Leu Ravine L4	Saint Leu Ravine L4

Table 1 (

 1

		Depth Sampling effort	Total 289
		Longitude
		Sampling date Latitude
		Cover b
		Probability of introduction a
		Most prob-	able lineage
		Stations
	continued)	Sites
		Zone

Table 2 Spearman

 2

	's matrix of correlations between	Pattern	Sites	Variables	Bioactivity	SST	PAR	NH 4 +	NO 3 -
	dependant (bioactivity) and independent variables (SST: sea surface temperature, PAR: photosynthetically active radiation, NH 4 + : ammonium concentration, NO 3 -: nitrate concentration, PO 4 3-: phosphate concentration)	Spatial Temporal	France	SST PAR SST PAR NH 4 + NO 3 -PO 4 3-	-0.428 (0.18) -0.370 (0.14) 0.287 (0.08) 0.175 (0.03) -0.373 (0.14) -0.435 (0.19) 0.079 (0.06)	0.377 0.661 -0.617 -0.654 -0.450	-0.622 -0.233 -0.294	0.811 0.640	0.602
			Réunion	SST	-0.
		Bold numbers show significant value at the level α ≤ 0.05. Coefficient of determination (Spearman R 2 ) are
		into brackets

729 (0.53) PAR -0.532 (0.28) 0.913 et

al. 2006b

). However, only few grazers are recognized to feed on Asparagopsis: the sea hare Aplysia parvula

(Paul