Matthieu Ghilain

Lise Hobeika

Micheline Lesaffre

Loris Schiaratura

Ashmita Singh

Joren Six

Dominique Huvent-Grelle

François Puisieux

Séverine Samson
email: severine.samson@univ-lille.fr

Does a live performance impact synchronization to musical rhythm in cognitively impaired elderly?

Keywords: Alzheimer's disease, Dementia, Music therapy, Social interaction, Motor activity, Aging, Cognitive impairment

Background

Music-based interventions appear to be efficient non-pharmacological approaches to improve emotional, social, and cognitive functioning of patients with neurodegenerative diseases such as Alzheimer's Disease (AD).

Objectives

Because the benefits seem to increase with patients motor involvement, we studied the ability to move in synchrony with musical rhythms and more specifically the impact of live performance compared to video recording of a musician on sensorimotor abilities in patients with cognitive impairments (AD, vascular and mixed dementia) and in patients with no evidence of cognitive impairments.

Methods

Sensorimotor synchronization (SMS) to a metronomic or a musical stimulus was assessed in patients when they were viewing a live musician or his pre-recorded video.

Results

Participants were better at synchronizing to a metronome than to music but this effect was modulated by the social context. While SMS to a metronome was better when facing a video than a live performance, there was no impact of the social context on SMS to music. Lastly, we found no group differences on SMS.

Conclusion

The decrease in synchronization ability to metronome in the live performance may be due to the social pressure associated with the presence of the musician. Such a pressure might be removed in pleasant and ecological social activities, like moving with music, explaining the lack of the social context effect on SMS to music. Remarkably, the group with cognitive impairments performed no worse than the group without cognitive impairments, which suggests relatively spared SMS abilities in these patients. By showing that it is possible to encourage a patient to synchronize with others, even in front of a video, the results of our study indicate that SMS can be used as a relevant predictor in clinical trials and open up promising therapeutic options for patients living in remote areas.

Introduction

Alzheimer's disease (AD) is a neurodegenerative disorder affecting several aspects of cognition, with symptoms such as memory dysfunction [START_REF] Hodges | Memory in the dementias[END_REF], language impairment [START_REF] Feyereisen | Pragmatic skills in the early stages of Alzheimer's disease : an analysis by means of a referential communication task[END_REF][START_REF] Potkins | Language impairment in dementia: Impact on symptoms and care needs in residential homes[END_REF], emotional deficits and social withdrawal [START_REF] Bediou | Impaired social cognition in mild alzheimer disease[END_REF]. Due to the limited efficacy of pharmacological treatments, health institutions have encouraged the development of nonpharmacological approaches in the management of dementia [START_REF]Dementia: Assessment, management and support for people living with dementia and their carers[END_REF]. In parallel, the use of music-based interventions for patients with AD and dementia has increased over the last decade [START_REF] Sihvonen | Music-based interventions in neurological rehabilitation[END_REF][START_REF] Van Der Steen | Music-based therapeutic interventions for people with dementia (Review)[END_REF] due to their positive impact on patients' emotional, social and cognitive functioning, as well as on caregiver well-being [START_REF] Narme | Efficacy of musical intervention in dementia: evidence from a randomized controlled trial[END_REF][START_REF] Choi | Effects of group music intervention on behavioral and psychological symptoms in patients with dementia: A pilot-controlled trial[END_REF]. Musical interventions can cover a large variety of activities from passive music listening in isolation to active music making in groups. Some studies reported that encouraging motor involvement in patients, by making them move with the musical beats or sing, provides greater benefits than passive music listening [START_REF] Sakamoto | Comparing the effects of different individualized music interventions for elderly individuals with severe dementia[END_REF][START_REF] Särkämö | Cognitive, emotional, and social benefits of regular musical activities in early dementia: Randomized controlled study[END_REF]. Such a finding suggests that motor coordination, and more precisely rhythmic sensorimotor synchronization (SMS), induced by these activities might contribute to the efficacy of music-based interventions in patients with dementia.

A central factor of these musical interventions is the social context: patients generally listen to music in the presence of others (i.e. in a group or in the presence of a musician). It has been demonstrated that the presence of a live musician while listening to music increases patients' spontaneous motor behaviors, compared with listening alone to an audio [START_REF] Sherratt | Music interventions for people with dementia: A review of the literature[END_REF][START_REF] Holmes | Keep music live: music and the alleviation of apathy in dementia subjects[END_REF][START_REF] Lesaffre | Monitoring music and movement interaction in people with dementia[END_REF] or a video [START_REF] Ghilain | Synchronisation sensorimotrice et comportements non verbaux dans la maladie d'Alzheimer : l'influence du contexte social et musical[END_REF] recording of a musical piece. Moreover, previous findings have shown that social presence can modulate SMS. When two individuals perform rhythmic movements together, they tend to spontaneously synchronize their movements [START_REF] Oullier | Social coordination dynamics : Measuring human bonding[END_REF][START_REF] Van Ulzen | Characteristics of instructed and uninstructed interpersonal coordination while walking side-by-side[END_REF]. By coupling their rhythmic movements with oneanother, individuals improve their SMS performances [START_REF] Demos | The unresponsive partner : roles of social status , auditory feedback , and animacy in coordination of joint music performance[END_REF][START_REF] Kirschner | Joint drumming: Social context facilitates synchronization in preschool children[END_REF][START_REF] Konvalinka | Follow you, follow me: continuous mutual prediction and adaptation in joint tapping[END_REF]. In a previous study, we investigated the impact of social context on SMS to musical rhythms as well as on patients' socio-emotional and motor engagement during musical activities in patients with AD [START_REF] Ghilain | Synchronisation sensorimotrice et comportements non verbaux dans la maladie d'Alzheimer : l'influence du contexte social et musical[END_REF]. Each participant was asked to tap with a metronomic or a musical rhythm, in the presence of a musician who performed the task. The presence of the musician was real (live condition) or virtual (video condition). Contrary to our predictions, we found that patients' SMS was better when they viewed a video of a musician performing rather than in a face-to-face live performance with a musician. We interpreted this finding by accounting for the effect of social pressure arising from the presence of an observer. Since the patients involved in our previous study all presented with moderate to severe dementia (Alzheimer's Disease, vascular or mixed dementia), and control participants without cognitive impairment were not tested, it remained difficult to determine whether or not the impact of social pressure was related to or exacerbated by the progression of the disease, and by cognitive decline. However, this issue deserves to be further investigated in elderly patients with different levels of cognitive impairments.

Rhythmic SMS requires core cognitive skills such as anticipation, adaptation and attention [START_REF] Repp | Sensorimotor synchronization: a review of recent research (2006-2012)[END_REF], as well as accurate sensorimotor control. Although spontaneous rhythmic movements are frequently observed, SMS has been rarely investigated in patients with neurodegenerative disease such as AD. Since most studies have explored deficits in cognition, little is known about the motor disorders associated with AD or other types of dementia [START_REF] Kluger | Motor/psychomotor dysfunction in normal aging, mild cognitive decline, and early alzheimer's disease: diagnostic and differential diagnostic features[END_REF][START_REF] Yan | Alzheimer's disease and mild cognitive impairment deteriorate fine movement control[END_REF]. The few studies that have examined rhythmic tapping abilities in AD have used continuous metronomic tapping tasks [START_REF] Martin | Human movement science alteration of rhythmic unimanual tapping and anti-phase bimanual coordination in Alzheimer's disease : a sign of inter-hemispheric disconnection ?[END_REF][START_REF] Bangert | Keep up the pace: declines in simple repetitive timing differentiate healthy aging from the earliest stages of Alzheimer's disease[END_REF]. In these motor tasks, which are more cognitively challenging than SMS tasks, patients start by tapping with metronome but are required to continue reproducing the metronome even after its removal. Results have shown a decrease in tapping regularity and accuracy in patients with AD, even at the early stage of the disease, as compared with matched elderly participants. However, the origins and the specificity of this decline in performance are not known.

When evaluating SMS, generally two types of sound sequences are used: metronome or musical sequences. Whereas the beat is directly perceived with a metronome, it has to be extracted from the complex auditory patterns in music. It is therefore harder to synchronize with music than with metronome beats [START_REF] Aschersleben | Temporal control of movements in sensorimotor synchronization[END_REF][START_REF] Bella | BAASTA : Battery for the Assessment of Auditory Sensorimotor and Timing Abilities[END_REF] even though tapping with music is more motivating and enjoyable. Additionally, SMS can be characterized by two complementary measures: consistency (i.e. tap regularity), and asynchrony (i.e. tap precision or anticipation). In the field of neurodegenerative pathologies, data reported in the literature has shown that SMS with music in patients with Parkinson's disease is less consistent and less anticipated than in controls [START_REF] Bienkiewicz | Parkinson's is time on your side ? Evidence for difficulties with sensorimotor synchronization[END_REF][START_REF] Cock Vc De | Rhythmic abilities and musical training in Parkinson's disease : do they help ?[END_REF][START_REF] Merchant | Interval timing and Parkinson's disease : heterogeneity in temporal performance[END_REF]. No study, to our knowledge, has compared SMS in patients with AD and in controls.

In line with our previous study, the present one aimed to investigate the impact of social context on SMS to musical rhythms in a different group of cognitively impaired elderly patients (CI group) and compare their performances to elderly participants with no cognitive impairments (NoCI group). The novelty of our paradigm lies in our ability to manipulate the social contexts, in which the task was performed. For this purpose, we used a simple paced tapping task, where participants synchronized tapping to beats in an auditory sequence. While performing the task, each participant viewed a musician who was also tapping synchronously to the auditory sequence, but this occurred in two distinct social environments. In one, participants were privy to a live performance by the musician, leading to face-to-face interactions (live condition). In the other condition, participants were shown the musician's pre-recorded performance projected on a life-sized screen (video condition). Furthermore, auditory context was manipulated by examining SMS in two types of auditory sequences: a metronomic and a musical sequence. These were analyzed based on the participants' consistency and asynchrony. We also measured the participants' spontaneous tapping in order to have an independent evaluation of their production abilities. For this task, participants were instructed to tap as regularly as possible at a comfortable rate. In line with previous findings, we expected SMS to be more consistent and more anticipated with metronome than with music, and in video than in live condition. We predicted that SMS in the CI group will be globally less consistent and anticipated than in the NoCI group; and consequently, based on the social cognition deficits associated with cognitively impaired elderly, we hypothesized that any interaction of social and auditory contexts may be more pronounced in participants with no cognitive impairment.

Materials and Methods

Participants.

Ninety-seven participants were recruited at the Bateliers Day Hospital (Lille University Hospital, France) in the context of their scheduled medical consultations (following memory or fall problems). All participants were right-handed, and native French speakers. A group of 48 patients with cognitive impairments (CI group) including patients with mild to moderate AD (n=12), patients with mixed dementia (n=30) and patients with vascular dementia,(n=6) participated in this study. The diagnosis of Alzheimer's disease (AD), vascular or mixed dementia was based on the Diagnostic and Statistical Manual of Mental Disorders-V criteria (American Psychiatric Association, 2013) and made by a geriatrician. Their results were compared to those of a group of 49 matched patients with no sign of cognitive impairments at the time of testing (NoCI group).

Demographic data encompassing age, gender, sociocultural level [START_REF] Poitrenaud | Evaluation du niveau socio-culturel[END_REF], musical expertise [START_REF] Ehrlé | Traitement temporel de l'information auditive et temporal[END_REF], as well as clinical information, including State-Trait Anxiety Inventory [START_REF] Spielberger | Manual for the state-trait anxiety inventory[END_REF], Activities of Daily Living [START_REF] Galasko | An Inventory to Assess Activities of Daily Living for Clinical Trials in Alzheimer's Disease[END_REF], and Mini-Mental State Examination [START_REF] Folstein | A practical state method for grading the cognitive state of patients for the clinician[END_REF] for both groups are presented in Table 1. The study was approved by the Comité de Protection des Personnes (CPP 18/012) and the Commission Nationale de l'Informatique et des Libertés (CNIL).

Apparatus

The experimental device consisted of two chairs facing each other. A tablet equipped with a force sensor was attached to the right armrest of each chair, allowing the participant and the musician to perform the SMS task by tapping on the tablet with a ball-like probe in their hands (Figure 1.A). Two social contexts were tested. In the video condition, the musician appeared in front of the participant on a life-size screen (158 x 92 cm) at a distance of 215 cm (Figure 1.B). In the live condition, the musician was facing the participant at a distance of 200 cm (Figure 1.C). The experimenter was hidden behind a curtain in both social contexts.

Two 60-second auditory sequences were used: a metronomic and a musical sequence, both presented at the same tempo (inter-onset interval (IOI) of 800ms, corresponding at 75 beats per minutes (bpm)). The metronomic sequence was composed of regular beats. The musical sequence was a musical excerpt from a French folk song ("La Java Bleue"), very well known to the elderly as attested by the participants themselves, with a ternary metric. Four initial beats preceded each auditory sequence to give the tempo.

Design and procedure

Each participant was tested individually. First, participants performed a spontaneous motor task, by tapping as regularly as possible for 30 taps at a comfortable tempo. Then, participants performed the SMS task by tapping at every beat for all the auditory sequences. The musician also tapped on his tablet at every beat of the auditory sequences, in both, live and video conditions. In addition, he pronounced "ta" at each beat of the metronome sequence and sang the lyrics of the song in the music sequence, with the exact same song excerpts being used during live and video conditions. The participant and the musician performed the SMS task together. The experimental design of the SMS task included one between-subject factor: GROUP (CI /NoCI); and two within-subject factors: AUDIO (Metronomic/Musical) and SOCIAL CONTEXT (Video/Live). The order of presentation of the four experimental conditions was counterbalanced.

Data analysis

The analysis of the spontaneous motor tapping was performed on 20 taps (the first eight taps and the last two taps being discarded). The mean tapping rate (mean intertap interval, ITI) and the tapping variability (coefficient of variation (CV) of the ITI) were calculated. SMS measures were analyzed on 64 beats, from a 60-second auditory sequence for each condition (providing 256 measures by participant), using circular statistics [START_REF] Fisher | Statistical analysis of circular data[END_REF] with the CircStat Matlab Toolbox [START_REF] Berens | CircStat: a MATLAB toolbox for circular statistics[END_REF], which is a robust tool to quantify SMS when there is not a perfect match between the number of taps and the pacing stimuli [START_REF] Kirschner | Joint drumming: Social context facilitates synchronization in preschool children[END_REF]. Thus, it is appropriate when participants tend to miss taps or to tap more than once in response to one beat.

Each tap was represented on a circular scale going from -180° to +180°, representing the 800 ms IOI (see Figure 2). The beat's occurrence corresponds to 0°: it is the time at which participant was expected to tap. Taps were placed on the circular scale depending on their time delay in relation with the beats. For example, a tap occurring 200 ms after a beat is represented by a unit vector with an angle of +90°, whereas a tap occurring 200 ms before a beat corresponds to a unit vector with an angle of -90°. All vectors were averaged to obtain a mean resultant vector R [START_REF] Fisher | Statistical analysis of circular data[END_REF][START_REF] Berens | CircStat: a MATLAB toolbox for circular statistics[END_REF] allowing for the calculation of synchronization consistency (i.e. regularity) and the accuracy. Vector R length indicated the consistency between 0 and 1, with 1 corresponding to perfect consistency (all taps occurred at the same delay to the beat) and 0 corresponding to an absence of synchronization (the taps were randomly distributed between the beats). Accuracy was calculated only if participant's taps were not randomly distributed. Random distribution of taps was tested using the Rayleigh test for circular uniformity [START_REF] Fisher | Statistical analysis of circular data[END_REF][START_REF] Wilkie | Rayleigh Test for Randomness of Circular Data[END_REF]. Accuracy was determined based on the angular deviation ('θ') of vector R from zero, which was then transformed back into milliseconds. The musician's synchronization performance was checked on the basis of the same 64 beats. The consistently good and proper performances by the musician did not justify the use of circular statistics. The accuracy and variability of synchronization were calculated by the mean and the CV of asynchronies expressed in milliseconds.

For each analysis, distribution normality was verified using the Kolmogorov-Smirnov test. Wilcoxon Mann-Whitney U t-test, a non-parametric test, was used in cases where the distribution was not normal. The Pearson's Chi-squared test was used for categorical variables (gender and socio-cultural level ratios). The sample effect size for ANOVA was estimated with the partial eta-squared (η 2 p).

Results

Preliminary analyses

Demographic and clinical data

We compared demographic and clinical data of the CI and NoCI groups. As summarized in Table 1, the analyses showed no group difference in terms of age (t(95) = 1.74, p = .08), gender (Chi-squared Pearson test : χ 2 (1, N = 97) = .18, p = .67), socio-cultural level (Chi-squared Pearson test : χ 2 (3, N = 97) = 3.38, p = .34), musical expertise (Mann-Whitney U test : U = 1075, p = .46), and mood assessed with the STAI mood questionnaire (Mann-Whitney U test : U = 930, p = 0.08). In contrast, the two groups differed in the severity of cognitive impairment as assessed by the MMSE (Mann-Whitney U test: U = 0, p <.001), and in functional status as assessed by ADLs (Mann-Whitney U test : U = 543, p <.001).

Spontaneous motor tapping task

Group comparison revealed no difference in ITIs and CV, indicating that pace and variability did not differ between groups (Mann-Whitney U test: p >.05 in both cases) during spontaneous motor tapping (see Figure 3).

Sensorimotor synchronization task

By using the Rayleigh test, we first verified whether participants tapped in synchrony with the beat or at random. All the tests were significant except for one NoCI participant who had a non-significant Rayleigh test in one condition, suggesting that this participant did not succeed at synchronizing the taps to the beat in this case. Since this occurred in only one condition for the participant and not in the other conditions, this specific value was replaced by the group average for this condition. SMS Consistency. We conducted an ANOVA on SMS consistency with the factors AUDIO (metronome/music), SOCIAL CONTEXT (video/live) and GROUP (CI/NoCI). Analysis revealed significant main effect of AUDIO (F (1,95) = 70.60, p <.001, η 2 p =.43), participants' taps being more consistent in metronome than in music conditions. There was also a significant main effect of SOCIAL CONTEXT (F (1,95) = 29.48, p <.001, η 2 p =.24), participants' taps being more consistent in video than in live conditions. Finally, there was a significant AUDIO by SOCIAL CONTEXT interaction (F (1, 95) = 32.44, p <.001, η 2 p =.25). When synchronizing with metronome beats, participants' taps were more constant in the video condition than in the live condition as confirmed by posthoc test with Bonferroni correction for multiple comparisons (p <.001), which was not the case when synchronizing with musical beats (see Figure 4.A). There was no significant GROUP effect or any other significant interactions (see Figure 4.C).

SMS Accuracy.

We conducted an ANOVA on SMS accuracy with the factors AUDIO (metronome/music), SOCIAL CONTEXT (video/live) and GROUP ((CI/NoCI). Analysis revealed significant main effects of the AUDIO (F (1,95) = 134.6, p <.001, η 2 p =.59), participants' taps being more anticipated in the metronome condition than in the music condition. There was also a significant main effect of the SOCIAL CONTEXT (F (1,95) = 18.09, p <.001, η 2 p =.16), participants' taps being more anticipated in the video condition than in the live condition. Finally they was a significant AUDIO by SOCIAL CONTEXT interaction (F (1,95) = 49.58, p <.001, η 2 p =.34). When synchronizing with metronome beats, participants' taps were more anticipated in the video condition than in the live condition, as confirmed by posthoc test with Bonferroni correction for multiple comparisons (p <.001), whereas when synchronizing with music (see Figure 4.B), the accuracy was not affected by the social context (p >.05) with taps being slightly delayed in both live and video conditions. There was no significant GROUP effect or any other significant interactions (see Figure 4

.D).

To verify whether or not singing during the music conditions interfered with SMS scores, we tested the correlations between duration of lip movements during singing and the two SMS measures (consistency and accuracy). The results showed no correlation in the live and video conditions with music (all ps >0.05) suggesting that singing activities did not disrupt SMS scores.

Musician's performances

We analyzed the musician's synchronization with the auditory sequences in the live condition. The musician's mean tapping rate (mean ITI) was significantly smaller (more synchronous) in the metronome than in the musical condition (Wilcoxon signed-rank test: Z = 8.21, p < .001), although his synchronization to the auditory sequences was highly accurate in both conditions (< 5 ms). Moreover, the musician's tapping variability (CV of the ITI) was significantly smaller in the metronome than the musical condition (Wilcoxon signed-rank test: Z = 7.43, p < .001). Table 2 indicates the musician's performances in the live and video conditions.

Discussion

The aim of this study was to evaluate the impact of the social context and of the auditory conditions on SMS abilities in two groups of elderly patients with or without cognitive impairments, at the time of testing. SMS was measured during a hand-tapping task in response to beats in metronomic or musical sequences, with a live or video presence of a musician. As expected, participants were better at synchronizing to metronome than to music with their taps being more consistent and more anticipated. We found that the effect of social context varied as a function of the auditory conditions. Although participants were better at synchronizing with a metronome beat when facing a pre-recorded video of a musician rather than a live performance, the social context had no effect on SMS in the musical condition. Furthermore, we did not observe differences between the two patient groups in any SMS scores.

Participants' SMS was more consistent and anticipated in the metronome than in the musical conditions. To ensure that these results cannot be explained by the musician's performance, we examined his SMS scores. As in the patients, they were better with the metronome than with the music. However, the difference between the musician's synchronization and the auditory beats in these two conditions was too small to explain the observed effect on patients' synchronization and to suggest that the musician adapted his performance to the patient's taps. Participants' decline in SMS performance in the musical condition reflects the complexity of synchronizing to beats in music. Indeed, SMS with a musical sequence requires extracting and anticipating the strong beats in a melody. Such synchronization is therefore more difficult to achieve than synchronization with a regular metronomic sequence composed exclusively of strong beats. This pattern of results, which has been documented in literature in healthy young adults [START_REF] Aschersleben | Temporal control of movements in sensorimotor synchronization[END_REF][START_REF] Repp | Sensorimotor synchronization : A review of the tapping literature[END_REF] and in individuals with Parkinson's disease [START_REF] Benoit | Musically cued gait-training improves both perceptual and motor timing in Parkinson ' s disease[END_REF], also extends to a general aged population. Interestingly, we found that SMS performances with metronome and music were differently impacted by the social context. In other words, when the participant tapped to a metronome in front of a live musician, consistency and anticipation decreased, as compared with a video of the musician. Such social effects were not obtained when participants synchronized taps to music. In the metronome conditions, participants' SMS performance from the present study was better when facing a video of a musician, rather a live presence. This result seems counterintuitive as studies on young adults support the idea that interpersonal interaction improves SMS with metronome [START_REF] Konvalinka | Follow you, follow me: continuous mutual prediction and adaptation in joint tapping[END_REF] and also with music [START_REF] Demos | The unresponsive partner : roles of social status , auditory feedback , and animacy in coordination of joint music performance[END_REF]. However, the present result is congruent with data we obtained in a group of patients with moderate to severe AD [START_REF] Ghilain | Synchronisation sensorimotrice et comportements non verbaux dans la maladie d'Alzheimer : l'influence du contexte social et musical[END_REF]. The deterioration in SMS with live performance may be explained due to the cognitive load of synchronizing to the auditory stimuli competing with that of synchronizing to the musician's movements [START_REF] Demos | Rocking to the beat: effects of music and partner's movements on spontaneous interpersonal coordination[END_REF]. However, this explanation is unlikely considering the high temporal precision and regularity with which the musician synchronized with the auditory sequences. An alternative explanation is that the SMS performance decreased in the live condition because of the mere presence of an observer. Audience effects refer to the social influence of having another individual present during a task [START_REF] Zajonc | Social facilitation[END_REF]. As reported in social psychology, the sole presence of an observer induces social pressure and stress, which can positively or negatively modulate an individual's performance in a task [START_REF] Welford | Stress and performance[END_REF][START_REF] Bray | Social facilitation among interacting groups: Evidence for the Evaluation-Apprehension Hypothesis[END_REF][START_REF] Mckinney | The Effects of Audience Size on High and Low Speech-Anxious Subjects During an Actual Speaking Task[END_REF]. In our study, we hypothesized that live presence of the musician may improve participants' performances by motivating them and by focusing their attention on the tapping task. On the contrary, it seems that live presence deteriorated participants' performances by dividing their attention. Whatever the underlying mechanism may be, increased cognitive load or existence of social pressure, the physical presence of another individual deteriorated performance in this task. The aforementioned effect was not dependent on participants' group, suggesting that this response pattern is not specific to the neurodegenerative diseases investigated here (AD, vascular and mixed dementia). Assessing participants' attention and social cognition (social anxiety, empathy) may allow a better understanding of this phenomenon in the future.

Unlike the metronome condition, the social context had no impact on participants SMS in the musical condition, the taps being delayed in response to the musical beat. Several hypotheses can be proposed to explain this difference. First, synchronizing with a musical beat is more difficult than synchronizing with a metronome one, and account for why SMS scores were globally less consistent and less anticipated (and even slightly delayed) in music condition. Tapping variability in the musical condition may have flattened the differences associated with social context. Second, music is a pleasant stimulus that can modulate the emotional state and increase social sharing of experiences. The stress induced by a live social presence, leading to performance deterioration in the metronome condition, may be mitigated when synchronizing to music due to its positive emotional impact. Furthermore, moving to music with another individual is a pleasant and ecological social task, close to a real life situation [START_REF] Wiltermuth | Synchrony and cooperation[END_REF][START_REF] Ehrenreich | Dancing in the streets: A history of collective joy[END_REF]. Thus, the musical condition might be more natural and enjoyable than the metronomic one, diminishing the audience effect.

In contrast to our expectations, we found no differences in rhythmic performances between the CI and the NoCI groups, be it in the spontaneous tapping task or in the SMS task. It is noteworthy that the spontaneous motor tempo (ITI of 800 ms or less, corresponding to 75bpm or more) obtained in these patients is consistent with data previously reported in patients with AD [START_REF] Rabinowitz | Association between finger tapping, attention, memory, and cognitive diagnosis in elderly patients[END_REF], and slower than the tempo generally observed in healthy elderly people [START_REF] Mcauley | The time of our lives : life span development of timing and event tracking[END_REF]. This result also confirmed that the tempo that we chose in this study for the SMS task (IOI of 800 ms, or 75bpm) was appropriate for this patient population. The lack of group difference in SMS performance may be slightly surprising for some, as previous studies have reported rhythmic deficits in AD using continuous tapping tasks [START_REF] Martin | Human movement science alteration of rhythmic unimanual tapping and anti-phase bimanual coordination in Alzheimer's disease : a sign of inter-hemispheric disconnection ?[END_REF][START_REF] Bangert | Keep up the pace: declines in simple repetitive timing differentiate healthy aging from the earliest stages of Alzheimer's disease[END_REF]. However, in these tasks, participants needed to continue reproducing a beat without the help of external stimuli, which is more cognitively demanding terms of attentional resources and working memory than the SMS task used in our study. Our present results suggest that SMS abilities are comparable in patients with or without cognitive impairments, despite a significant cognitive and behavioral decline in the CI group. As evidenced by neuroimaging studies [START_REF] King | Increased Functional Connectivity After Listening to Favored Music in Adults With Alzheimer Dementia[END_REF][START_REF] Jacobsen | Why musical memory can be preserved in advanced Alzheimer ' s disease[END_REF][START_REF] Zatorre | When the brain plays music: Auditory-motor interactions in music perception and production[END_REF], the neural correlates of synchronization and audio-motor coupling include the pre-motor cortex, the pre-supplementary motor area, the basal ganglia and the cerebellum. It might therefore be possible that these regions are relatively preserved even in moderate AD; however we approach this interpretation cautiously since about three quarters of the patients in the CI group presented a mixed or a vascular dementia with different cerebral dysfunction. Even though the participants of the CI group were impaired as compared to participants of the NoCI group in terms of cognitive (MMSE [START_REF] Folstein | A practical state method for grading the cognitive state of patients for the clinician[END_REF]) and behavioral measures (ADL score [START_REF] Galasko | An Inventory to Assess Activities of Daily Living for Clinical Trials in Alzheimer's Disease[END_REF]), we cannot be sure that patients in the NoCI group were all healthy, given that for many the hospital visit was instigated by a recent fall. Motor difficulties and particularly those related to walking and falls can be a predictor for the onset of Parkinson's disease and even AD [START_REF] Belghali | Loss of gait control assessed by cognitive-motor dualtasks : pros and cons in detecting people at risk of developing Alzheimer ' s and Parkinson ' s diseases[END_REF][START_REF] Buracchio | The Trajectory of Gait Speed Preceding Mild Cognitive Impairment[END_REF][START_REF] Verghese | Quantitative gait dysfunction and risk of cognitive decline and dementia[END_REF]. We have therefore no guarantee that the NoCI group were not at a prodromal stage of a degenerative disease. Nevertheless, we can be quite confident that these matched participants from the NoCI group presented fewer cognitive and behavioral deficits in comparison with patients from the CI group. To better document this question, further research needs to be carried out to evaluate SMS abilities in healthy older adults but also in patients at several time-points during their disease progression.

To conclude, our results show that it is possible to collect well-controlled measurements of SMS in elderly people with or without cognitive deficits. Although experimental rigor is often difficult to reconcile with the constraints of clinical reality, particularly in the field of pathological ageing, this experimental paradigm based on a hand-tapping task performed with a partner as part of a musical task, seems suitable for evaluating SMS. Thus, this study validates an experimental device consisting of a chair equipped with a tablet to record SMS in patients with impaired cognitive faculties (decreased attention, motor, motivation and communication disorders). This paradigm is also original in its examination of the impact of social interaction induced by a live partner compared to a virtual presence. By showing that it is possible to encourage a patient to synchronize with others, even in the presence of a video recording, our study provides a relevant measure to predict clinical effects that might used as outcome measures in future studies. It also opens up promising therapeutic potential for patients living at home, in remote areas or in confinement. Mobile technology might be used to train patients. By synchronizing movements to music and engaging patients in rhythmic action with others, cognitive-motor skills and temporal tracking mechanisms involved in verbal and non-verbal interaction would be stimulated. Stimulating SMS and musical entrainment in patients might increase the alignment of internal oscillations between brain regions with that of beats in auditory rhythms. By progressively acting in synchrony with an environmental system (musical rhythm) through the coupling of perception and action, we propose that cognition and social abilities will improve through the use of rhythmic stimulation in elderly. We therefore predict that such trainings will better the action-perception coupling and interpersonal coordination, consequently boosting verbal and non-verbal communicative skills. This remains to be verified in the future. The idea is not to replace musical activities performed in the presence of others with a virtual interaction, but rather to complement classical interventions. Virtual initiatives, via tele-health and similar platforms, are an unexplored cost-effective and easy to implement tool to address everyday social isolation experienced by this geriatric population. Being able to employ video recordings and mobile technology may allow healthcare providers to circumvent practical difficulties (i.e. rural and low-resource settings, or other exceptional situation such as the current pandemic) that may act as a barrier to providing care that has historically been considered niche or resource intensive. and A. Clerckx, to the staff of the Bateliers day hospital at Lille, and to the participants who agreed to participate in this study. was better with more anticipation when synchronizing with a metronome beat than a musical one. Moreover, when participants tapped with metronome beats, their consistency was better (panel A) and their anticipation larger (panel B) with a video presence than with a live presence of a partner. When participants tapped with musical beats, the social context did not impact their consistency and accuracy. No significant difference was observed between the CI and the NoCI groups on the consistency (panel C) and the accuracy (panel D). n.s.= non-significant

Figure 1 :Figure 2 .

 12 Figure 1: Experimental set-up. (A) Participant performed the SMS task in front of a musician, by tapping on a tablet fixed on the chair right armrest. The musician was present either as (B) a life size recorded broadcast on a screen (video condition), or (C) a live performance (live condition)

Figure 3 :

 3 Figure 3: Spontaneous tapping task. This figure reports the mean inter-tap interval (ITI) (± SEM) and the mean coefficient of variation (CV) (± SEM) of the CI group and the NoCI group. No significant difference was found between groups. n.s.= non-significant

Figure 4 :

 4 Figure 4: Analysis of SMS consistency and accuracy. This figure reports mean synchronization consistency (±SEM) and accuracy (±SEM) depending on the auditory sequence (metronome or music) and the social context (video or live) (panel A and B), and depending on the participant group (panel C and D). Consistency can vary between 0 (random distribution of taps) and 1 (perfect regularity of taps). Negative accuracy values indicate taps occurring before the beat (anticipated), whereas positive accuracy values indicate taps occurring after the beat (delayed). Participants' consistencywas better with more anticipation when synchronizing with a metronome beat than a musical one. Moreover, when participants tapped with metronome beats, their consistency was better (panel A) and their anticipation larger (panel B) with a video presence than with a live presence of a partner. When participants tapped with musical beats, the social context did not impact their consistency and accuracy. No significant difference was observed between the CI and the NoCI groups on the consistency (panel C) and the accuracy (panel D). n.s.= non-significant

Table 1 .

 1 Demographic and clinical characteristics (mean ± std) of patients with cognitive impairments (CI) and patients without cognitive impairment (NoCI). Socio-cultural level evaluated according to 4 levels. Level 1 is the lowest, corresponding to the absence of diploma. Level 4 is the highest, corresponding to a high school diploma b missing data for one participant

	CI (N = 48)	NoCI (N = 49)	p

a

Table 2 .

 2 Musician's performances. Mean hand-tapping rate (mean inter-tap interval, ITI) (±SEM) and mean tapping variability (coefficient of variation (CV) of the ITI) (±SEM) of the musician in both auditory (metronome/musical) and social conditions (video/live).

			Inter-tap interval (ITI)	Coefficient of variation (CV)
			(ms)	(ms)
	Metronome Video	-.13	.30
		Live	.57 ± .15	.90 ± .031
	Music	Video	3.81	1.1
		Live	4.35. ± .16	1.1 ± .18

Acknowledgements

This research was carried out with the support of the Conseil Régional des Hauts-de-France and the University of Lille at M.G., the Ministry of Foreign Affairs (Hubert Curien partnership) at S.S. and M.L. as well as the France Alzheimer and Institut Universitaire de France at S.S. This research was carried out in cooperation with the Department of Musicology of Ghent University, and financed by the project "Expressive Music Interaction" which is financially supported by the Flemish government (Methusalem grant). This research was also supported by the Globalink Research Award from Mitacs Canada to A.S. The authors would also like to thank Ivan Schepers of Ghent University for the development of the material. We are especially grateful to Sotirios Sideris, the musician who participated in the study, to the geriatrician Dr. J. Roche, to the psychologists C. Jougleux, S. Schoenenburg

Acknowledgements

The authors would like to thank Ivan Schepers of Ghent University for the material development. We are especially grateful to the musician who participated in the study Sotirios Sideris, to the geriatrician Dr. J. Roche, to the psychologists C. Jougleux, S. Schoenenburg and A. Clerckx, to the staff of the Bateliers day hospital at Lille, and to the participants who agreed to participate in this study.

Conflict of interest

The authors have no conflict of interest to report.


Guinand Bruno

Bruno Guinand

Münevver Oral

Christelle Tougard

Brown trout phylogenetics: a persistent mirage towards (too) many species

… such abundant literature and insightful studies may be little more than a mirage" J

understanding in both the phylogeny of the Salmo genus and the phylogeny of the brown trout, thus in their origin, diversification and other aspects of their evolutionary history. The brown trout complex received an interest not found in any other (Pan-)European fish 'species' so far. Indeed, outside population genetics issues that target one single 'species' or -generally speaking -a single operational taxonomic unit (OTU), a literature review estimated the number of original phylogeographic and phylogenetic studies considering at least two OTUs -clades, lineages or (sub)species -to approx. 130 (Fig. 1). Such interest should have led to significant improvements of knowledge, including taxonomic progress.

While a review of knowledge based also on over 100 studies has been already provided by [START_REF] Sanz | Phylogeography history of brown trout: A review[END_REF], we however believe we are facing a mirage of extensive knowledge in trout. If we do not oppose to contents of her work, a difference between [START_REF] Sanz | Phylogeography history of brown trout: A review[END_REF] review and this opinion piece could be framed in the well-known 'splitter-lumper' dichotomy. Hereby, our goal is to be 'splitters' in order to stress that the genealogical relationships resulting from speciation within the brown trout complex remains unfortunately neglected. Otherwise, works as summarized by [START_REF] Sanz | Phylogeography history of brown trout: A review[END_REF] rather depict 'lumping' in which most molecular data produced to help for the delimitation of trout species or another taxonomic entity are aggregated to one existing reference, S. trutta sensu stricto (hereafter, StSS) whose origin is offered below. This opinion piece hence suggests to reconsider the outcomes and to come back on some issues that initially motivated trout molecular studies, i.e. providing a sound phylogenetic frame and the evolutionary history of described taxa, before to establish, redefine or improve -or not -operational criteria necessary to species delimitation.

Thirty years of research in few lines: what is really known?

If former allozyme studies anticipated the rise of (mt)DNA studies in trout and identified phylogenetic/phylogeographic signals (reviews in [START_REF] Ferguson | Genetic differences among brown trout, Salmo trutta, stocks and their importance for the conservation and management of the species[END_REF][START_REF] Guyomard | Diversité génétique de la truite commune[END_REF][START_REF] García-Marín | Post-glacial colonization of brown trout in Europe based on distribution of allozyme variants[END_REF], works by [START_REF] Bernatchez | DNA sequence variation of the mitochondrial control region among geographically and morphologically remote European brown trout Salmo trutta populations[END_REF], [START_REF] Bernatchez | The evolutionary history of brown trout (Salmo trutta L.) inferred from phylogeographic, nested clade, and mismatch analyses of mitochondrial DNA variation[END_REF][START_REF] Bernatchez | Genetic diversity of trout (genus Salmo) from its most eastern native range based on mitochondrial DNA and nuclear gene variation[END_REF][START_REF] Bernatchez | The evolutionary history of brown trout (Salmo trutta L.) inferred from phylogeographic, nested clade, and mismatch analyses of mitochondrial DNA variation[END_REF] represent the main first attempts to investigate trout diversity using sequencing molecular tools in a Pan-European perspective. Since these works, main observations might be briefly summarized:

(1) The five original mtDNA StSS lineages reported in [START_REF] Bernatchez | DNA sequence variation of the mitochondrial control region among geographically and morphologically remote European brown trout Salmo trutta populations[END_REF] and [START_REF] Bernatchez | The evolutionary history of brown trout (Salmo trutta L.) inferred from phylogeographic, nested clade, and mismatch analyses of mitochondrial DNA variation[END_REF] -namely Atlantic, Danubian, Mediterranean, Adriatic and marmoratus (Fig. 2) -still anchored most studies in the field, but, their respective distribution area became more complex, resulting in the co-occurrence of mtDNA clades in some areas (e.g., Italy, Corsica, Balkans) rather than their allopatric distribution, as initially described [START_REF] Sanz | Phylogeography history of brown trout: A review[END_REF] for review);

(2) Few other StSS lineages have been added to the original ones in Spain (Duero: [START_REF] Suárez | Evolution of the mitochondrial control region in Palaearctic brown trout (Salmo trutta) populations: the biogeographical role of the Iberian Peninsula[END_REF], Turkey (Tigris: [START_REF] Bardakci | Phylogeography of the Turkish brown trout, Salmo trutta L.: mitochondrial DNA PCR RFLP variation[END_REF][START_REF] Sušnik | Genetic verification of native brown trout from the Persian Gulf (Catak Cay River, Tigris basin)[END_REF], Morocco (Dades; [START_REF] Snoj | Phylogeographic structure and demographic patterns of brown trout in North-West Africa[END_REF] and Northern Africa [START_REF] Tougard | Salmo macrostigma (Teleostei, Salmonidae): Nothing more than a brown trout (S. trutta) lineage?[END_REF] (Fig. 2). Sublineages related to the Atlantic lineage have been reported in Central Europe (e.g. [START_REF] Cortey | Northern and Southern expansions of Atlantic brown trout (Salmo trutta) populations during the Pleistocene[END_REF];

(3) Some species within the complex have lost their initial status and now related to one of the original StSS clade [START_REF] Kalayci | Genetic and molecular evidence that brown trout Salmo trutta belonging to the Danubian lineage are a single biological species[END_REF][START_REF] Mckeown | Phylogeographic structure of brown trout Salmo trutta in Britain and Ireland: glacial refugia, postglacial colonization and origins of sympatric populations[END_REF][START_REF] Sanz | Phylogeography history of brown trout: A review[END_REF][START_REF] Snoj | Resolving taxonomic uncertainties using molecular systematics: Salmo dentex and the Balkan trout community[END_REF]Sušnik et al., 2007a;[START_REF] Tougard | Salmo macrostigma (Teleostei, Salmonidae): Nothing more than a brown trout (S. trutta) lineage?[END_REF]. High rate of invalid taxa is not surprising in trout [START_REF] Jonsson | Ecology of Atlantic Salmon and Brown trout: Habitat as a Template for Life Histories[END_REF], and in salmonids in general (e.g. [START_REF] Adams | Arctic charr in Britain and Ireland -15 species or one?[END_REF]. However, the problem goes beyond taxon names and affects the understanding of evolutionary history. For example, Lobón-Cerviá (2018) reports a possible synonymy between S. macrostigma and S. cettii, when [START_REF] Tougard | Salmo macrostigma (Teleostei, Salmonidae): Nothing more than a brown trout (S. trutta) lineage?[END_REF] questioned the reality of S. macrostigma. Authors proposed S. cettii as belonging to the Adriatic StSS (e.g., [START_REF] Gratton | The evolutionary jigsaw puzzle of the surviving trout (Salmo trutta L. complex) diversity in the Italian region. A multilocus Bayesian approach[END_REF], while [START_REF] Tougard | Salmo macrostigma (Teleostei, Salmonidae): Nothing more than a brown trout (S. trutta) lineage?[END_REF] linked S. macrostigma to the North African StSS (Fig. 2). One or two species? One or two evolutionary histories? What is/are the realised distribution(s)? Confusion reigns. Furthermore, some OTUs should be hybrids [START_REF] Razpet | Genetic variation among trout in the River Neretva basin, Bosnia and Herzegovina[END_REF][START_REF] Mckeown | Phylogeographic structure of brown trout Salmo trutta in Britain and Ireland: glacial refugia, postglacial colonization and origins of sympatric populations[END_REF][START_REF] Gratton | The evolutionary jigsaw puzzle of the surviving trout (Salmo trutta L. complex) diversity in the Italian region. A multilocus Bayesian approach[END_REF]. Otherwise, mtDNA data confirmed the classification of some species outside StSS: S. obtusirostris restricted to the western Balkans [START_REF] Snoj | DNA phylogeny supports revised classification of Salmothymus obtusirostris[END_REF] and S. ohridanus endemic to Lake Ohrid [START_REF] Phillips | Phylogenetic analysis of mitochondrial and nuclear sequences supports inclusion of Acantholingua ohridana in the genus Salmo[END_REF]Sušnik et al., 2006), with additional support coming from nuclear data (e.g. [START_REF] Pustovrh | Molecular phylogeny of Salmo of the western Balkans, based upon multiple nuclear loci[END_REF][START_REF] Lecaudey | Inferring phylogenetic structure, hybridization and divergence times within Salmoninae (Teleostei: Salmonidae) using RAD-sequencing[END_REF]. A proposal to erect the marmoratus lineage as a species outside StSS has been made by [START_REF] Pustovrh | Molecular phylogeny of Salmo of the western Balkans, based upon multiple nuclear loci[END_REF] based on nuclear data, but not thoroughly validated so far.

(4) One origin in the Middle East or/and Mesopotamia is postulated with a probable colonization of northern Europe by the Caspian and the Black Sea, and a colonization of Southern Europe following a Mediterranean route, southern of the Anatolian plateau (Bardakci et al., 2006 and references therein). This point is probable and evidence also comes from comparisons with other species groups (e.g. Squalius and Chondrostoma; [START_REF] Durand | Phylogeny and biogeography of the family Cyprinidae in the Middle East inferred from cytochrome b DNA -evolutionary significance of this region[END_REF][START_REF] Durand | Insight into the origin of endemic Mediterranean ichthyofauna: phylogeography of Chondrostoma genus (Teleostei, Cyprinidae)[END_REF]. The phylogeny presented in Fig. 2 based on StSS mtDNA haplotypes captured little of such information, placing in this case all the western European lineages (Duero, Atlantic and North African) at the root of the mtDNA brown trout phylogeny. It thus emphasizes one Atlantic refuge/Western European for European fish (e.g. [START_REF] Bryja | Range-wide population genetic structure of the European bitterling (Rhodeus amarus) based on microsatellite and mitochondrial DNA analysis[END_REF][START_REF] Culling | European colonization by the spined loach (Cobitis taenia) from Ponto-Caspian refugia based on mitochondrial DNA variation[END_REF][START_REF] Durand | Phylogeography and postglacial dispersion of the chub (Leuciscus cephalus) in Europe[END_REF] that has been discussed in trout (e.g. Sanz 2018) and Atlantic salmon (S. salar; e.g., [START_REF] Finnegan | Use of multiple markers demonstrates a cryptic western refugium and postglacial colonisation routes of Atlantic salmon (Salmo salar L.) in Northwest Europe[END_REF]. A refuge is however not an origin. MtDNA taxonomic frames provide interesting but incomplete versions of evolutionary histories [START_REF] Dellicour | The hitchhiker's guide to single-locus species delimitation[END_REF][START_REF] Toews | The biogeography of mitochondrial and nuclear discordance in animals[END_REF], and no Pan-European nuclear-DNA study is available in trout so far. [START_REF] Sanz | Phylogeography history of brown trout: A review[END_REF] reviewed and proposed mtDNA estimates for divergence time among S. ohridanus-S. obtusirostris and S. trutta that rarely exceed 2 Myr (i.e. Early Pleistocene, concordant with the oldest fossil attributed to S. trutta ;[START_REF] Vladimirov | The trout from the Diatomite deposits of the Pliocene[END_REF].

However, recent fossil-calibrated, nuclear estimates provided divergence times around 4-6 Myr between brown trout and S. ohridanus-S. obtusirostris, and divergence with Atlantic salmon dating back from the Middle Miocene (15-10 Myr) [START_REF] Crête-Lafrenière | Framing the Salmonidae family phylogenetic portrait: A more complete picture from increased taxon sampling[END_REF][START_REF] Lecaudey | Inferring phylogenetic structure, hybridization and divergence times within Salmoninae (Teleostei: Salmonidae) using RAD-sequencing[END_REF]. Unfortunately, for both mtDNA and nuclear estimates, this did not extend to other OTUs than those reported in Fig. 2.

(5) Finally, if we emphasize the lack of updated molecular phylogeny encompassing most proposed trout taxa in this piece, no complete morphology-based phylogeny has been proposed either. Only partial attempts including few taxa have been produced (e.g., [START_REF] Salmanov | Comparative osteology of river and lake trouts (genus Salmo)[END_REF]. Correlations between molecular and morphological observations supporting taxonomy resulted in mixed results (see [START_REF] Sanz | Phylogeography history of brown trout: A review[END_REF][START_REF] Delling | Morphologic and genetic characterisation of Corsican and Sardinian trout with comments on Salmo taxonomy[END_REF]. Further investigations are requested to understand the molecular underpinnings of observed phenotypic and life-history differences that led to trout biocomplexity. To date, only [START_REF] Jacobs | The genetic architecture underlying the evolution of a rare piscivorous life history form in brown trout after secondary contact and strong introgression[END_REF] really addressed this issue at a genomic scale for the S. trutta/S. ferox 'species' pair.

The initial frame that focused on StSS is thus valid, now better evaluated, and it has been enriched by additional lineages and better delineation of relationships to few other taxa. It however remains mostly dependent on uniparentally inherited mtDNA data (Fig. 1).

Overall, the number of supported taxa and more importantly their relationships, their rate and patterns of diversification, their colonization histories, and their links to phenotypic diversification that support taxonomic descriptions remain obscure. Deep knowledge is a mirage and we may question if scientists built a complex to not face their icon.

Two more frames

If a coherent phylogenetic frame is still missing, two other frames strongly cohabit in trout molecular studies: one concerns species definition, taxonomy and their associated revisions (supra and Fig. 2), while a second is sounded in conservation issues -often crucial in trout -and should be 'taxonomy independent' [START_REF] Splendiani | The name of the trout: considerations on the taxonomic status of the Salmo trutta L., 1758 complex (Osteichthyes: Salmonidae) in Italy[END_REF]; see also [START_REF] Stanton | More grist for the mill? Species delimitation in the genomic era and its implications for conservation[END_REF]. These two frames are generally considered fundamental to design efficient biodiversity conservation priorities and management strategies [START_REF] Allendorf | Units of conservation[END_REF][START_REF] Eizaguirre | Evolutionary conservation -evaluating the adaptive potential of species[END_REF]), but their uncoupling has been promoted by some agencies (e.g. [START_REF] Haig | Taxonomic considerations in listing subspecies under the U.S. Endangered Species Act[END_REF]. Briefly said, operational criteria to species delimitation should not be confused with operational criteria for conservation (e.g. [START_REF] Raposo | On the notions of taxonomic 'impediment', 'gap', 'inflation' and 'anarchy', and their effects on the field of conservation[END_REF]. In trout, the inappropriateness of considering the "old" zoogeographic frame for conservation and the preservation of its evolutionary diversity has been already discussed [START_REF] Antunes | Complex evolutionary history in the brown trout: insights on the recognition of conservation units[END_REF][START_REF] Apostolidis | Genetic divergence among native trout Salmo trutta populations from southern Balkans based on mitochondrial DNA and microsatellite variation[END_REF][START_REF] Mckeown | Phylogeographic structure of brown trout Salmo trutta in Britain and Ireland: glacial refugia, postglacial colonization and origins of sympatric populations[END_REF][START_REF] Schenekar | Fine-scale phylogeographic contact zone in Austrian Brown trout Salmo trutta reveals multiple waves of post-glacial colonization and predominance of natural vs anthropogenic admixture[END_REF]. Its use remains unfortunately pregnant while focus on local (meta)populations could be advertised to prioritize issues that are prompted to more efficient management and deal with biological characteristics, ecological and/or evolutionary potentials [START_REF] Antunes | Complex evolutionary history in the brown trout: insights on the recognition of conservation units[END_REF][START_REF] Splendiani | The name of the trout: considerations on the taxonomic status of the Salmo trutta L., 1758 complex (Osteichthyes: Salmonidae) in Italy[END_REF]. However, because species is the only recognized unit to frame conservation policies and because rare and endemic species rather than widely distributed ones are favoured, the two frames remain unfortunately lumped in trout by putting the species status or the lineage at the conservation forefront. Numerous trout studies thus focused on events occurring at temporal and/or spatial scales that are poorly relevant to conservation management and quite descriptive at the level of OTUs (e.g. updated distribution of haplotypes pointing new records or levels of diversity that are not related to life history or ecological data).

Other approaches that consider "the best of all worlds" might be substituted (e.g., [START_REF] Mee | Identifying designatable units for intraspecific conservation prioritization: a hierarchical approach applied to the lake whitefish species complex (Coregonus spp.)[END_REF]. Pillars of sands should be avoided, and, by abusively mixing the two frames, the goal to address the big phylogenetic picture, to reduce taxonomic uncertainty and to better understand the processes that shaped the brown trout complex has been progressively lost. This big picture has perhaps become still more difficult to address as new putative trout species have been described over the last fifteen years, notably in Morocco [START_REF] Delling | Systematics of the trouts endemic to Moroccan lakes, with description of a new species (Teleostei: Salmonidae)[END_REF][START_REF] Doadrio | Two new species of Atlantic trout (Actinopterygii, Salmonidae) from Morocco[END_REF] and the Anatolian and Taurus regions, including Tigris and Euphrates drainages (e.g., [START_REF] Turan | Salmo tigridis, a new species of trout from Tigris River, Turkey (Teleostei: Salmonidae)[END_REF][START_REF] Turan | Salmo kottelati, a new species of trout from Alakır Stream, draining to the Mediterranean in southern Anatolia, Turkey (Teleostei, Salmonidae)[END_REF][START_REF] Turan | Salmo munzuricus, a new species of trout from the Euphrates River drainage, Turkey (Teleostei: Salmonidae)[END_REF][START_REF] Turan | A new species of trout from the northern drainages of Euphrates River, Turkey (Salmoniformes: Salmonidae)[END_REF]. These are areas where novel StSS lineages were described (Dades, Tigris, see above), and perhaps close to the putative centre of origin of brown trout for Anatolian trout.

We thus do not currently know how these taxa are related, if they clustered within or outside StSS (could be considered as valid taxa or not, sensu [START_REF] Galtier | Delineating species in the speciation continuum: A proposal[END_REF], and which portion of trout evolutionary history they may allow to better understand.

The future

The next step in trout study is to handle the full complexity of phylogenetic relationships to learn how trout diversity arises and changes. Picking up only previously described 'species' one by one for further molecular comparison with StSS has become a dead-end. After decades of studies using maternally inherited mtDNA markers or -more generally -reduced marker sets, "going large" with next generation sequencing (NGS) technologies that provide access to thousands of nuclear loci (e.g. single nucleotide polymorphisms [SNPs]) and genomic features (e.g., [START_REF] Wellenreuther | Going beyond SNPs: The role of structural genomic variants in adaptive evolution and species diversification[END_REF] to delineate the underlying genealogy is undoubtedly the key in trout. Panels of genome-wide distributed SNP markers have proved suitable for phylogenomic analysis [START_REF] Leaché | The utility of single nucleotide polymorphism (SNP) data in phylogenetics[END_REF] and updated the understanding about the patterns and the mechanisms of lineage diversification, together with species delimitation (e.g., [START_REF] Chan | Gene flow creates a mirage of cryptic species in a Southeast Asian spotted stream frog complex[END_REF][START_REF] Díaz-Arce | RAD-seq derived genome-wide nuclear markers resolve the phylogeny of tunas[END_REF][START_REF] Hughes | Biogeography, habitat transitions and hybridization in a radiation of South American silverside fishes revealed by mitochondrial and genomic RAD data[END_REF][START_REF] Leaché | Species delimitation using genome-wide SNP data[END_REF][START_REF] Song | Species delimitation and phylogenetic reconstruction of the sinipercids (Perciformes: Sinipercidae) based on target enrichment of thousands of nuclear coding sequences[END_REF]. This includes fish species complex in sticklebacks [START_REF] Guo | A phylogenomic perspective on diversity, hybridization and evolutionary affinities in the stickleback genus Pungitius[END_REF], roaches [START_REF] Baumsteiger | Genomics clarifies taxonomic boundaries in a difficult species complex[END_REF], cichlids (e.g. [START_REF] Willis | no. Or, arbitrary assignment of lineages to species obscures the diversification processes of Neotropical fishes[END_REF]; suckers [START_REF] Bangs | Gene flow and species delimitation in fishes of Western North America: Flannelmouth (Catostomus latipinnis) and bluehead sucker (C. Pantosteus discobolus)[END_REF], topminnows [START_REF] Duvernell | Late Pleistocene range expansion of North American top minnows accompanied by admixture and introgression[END_REF], and coregonids (Coregonus ardeti species flock; [START_REF] Ackiss | Genotyping-by-sequencing illuminates high levels of divergence among sympatric forms of coregonines in the Laurentian Great Lakes[END_REF]. Based on several thousands of polymorphisms, [START_REF] Copus | Geopolitical species revisited: genomic and morphological data indicate that the roundtail chub Gila robusta species complex (Teleostei, Cyprinidae) is a single species[END_REF] proposed to reduce the Gila robusta complex to a single species. NGS data also provided with better assessment of origin, colonization routes, demographic histories including admixture and introgression, and/or identification of stage of divergence during the speciation process in diverse fish species (e.g., [START_REF] Jeffries | Comparing RADseq and microsatellites to infer complex phylogeographic patterns, an empirical perspective in the Crucian carp, Carassius carassius[END_REF][START_REF] Fang | Worldwide phylogeny of threespined sticklebacks[END_REF][START_REF] Lucek | Distinct colonization waves underlie the diversification of the freshwater sculpin (Cottus gobio) in the Central European Alpine region[END_REF][START_REF] Ravinet | The genomic landscape at a late stage of stickleback speciation: High genomic divergence interspersed by small localized regions of introgression[END_REF][START_REF] Rougemont | The demographic history of Atlantic salmon (Salmo salar) across its distribution range reconstructed from approximate Bayesian computations[END_REF]. For example, the pattern of reticulate evolution between S. obtusirostris and the Adriatic clade of S. trutta described by [START_REF] Sušnik | Reticulate evolution: ancient introgression of the Adriatic brown trout mtDNA in softmouth trout Salmo obtusirostris (Teleostei: Salmonidae)[END_REF] should receive in depth evaluation. Trout, however, covers perhaps more putative OTUs than any other examples, and OTU-rich taxa appeared yet especially challenging to investigate these issues (e.g. [START_REF] Dincă | The conundrum of species delimitation: a genomic perspective on a mitogenetically super-variable butterfly[END_REF][START_REF] Chambers | The multispecies coalescent over-splits species in the case of geographically widespread taxa[END_REF]. Actually, it is recognized that a limited number of sampled genomes per OTU may provide access to substantial information, including "evolutionary process connectivity" (i.e. how contemporary observed connectivity and diversity reflect long term selective/adaptive and demographic history, including patterns of genetic admixture and speciation; Gagnaire, 2020). The ever-decreasing cost to access genome data would probably no longer constrains this issue.

The release of important genomic resources such as the Atlantic salmon [START_REF] Lien | The Atlantic salmon genome provides insights into rediploidization[END_REF]GenBank: GCA_000233375.4) and brown trout (GenBank: GCA_901001165.1) genomes provide substrates to progress in trout phylogeny. SNPs are accessible in S.

trutta, but mostly used however in a population genomics framework (e.g., [START_REF] Andersson | Complex genetic diversity patterns of cryptic, sympatric brown trout (Salmo trutta) populations in tiny mountain lakes[END_REF][START_REF] Drywa | Genotyping of two populations of Southern Baltic Sea trout Salmo trutta m. trutta using an Atlantic salmon derived SNP-array[END_REF][START_REF] Leitwein | Genome-wide nucleotide diversity of hatchery-reared Atlantic and Mediterranean strains of brown trout Salmo trutta compared to wild Mediterranean populations[END_REF][START_REF] Saint-Pé | Development of a large SNPs resource and a low-density SNP array for brown trout (Salmo trutta) population genetics[END_REF]. Jacobs et al. The production of a more complete phylogeny including many taxa seem possible, but challenges have to be overcome. This includes the relatively young history of divergence for some OTUs, taxon sampling, choosing marker types, or identification of orthologous/paralogous genes (single-copy vs gene duplicates) in trout. As paralogs impede investigations in salmonids [START_REF] Lecaudey | Inferring phylogenetic structure, hybridization and divergence times within Salmoninae (Teleostei: Salmonidae) using RAD-sequencing[END_REF], databases [START_REF] Pasquier | Gene evolution and gene expression after whole genome duplication in fish: the PhyloFish database[END_REF][START_REF] Samy | SalmoBase: an integrated molecular data resource for Salmonid species[END_REF] and tools providing guidelines for de novo or graph-based inference methods for orthologous gene identification have been developed [START_REF] Kapli | Phylogenetic tree building in the genomic age[END_REF][START_REF] Smith | New approaches for inferring phylogenies in the presence of paralogs[END_REF]. Many of them seem however already identified owing to transcriptome assembly in the brown trout [START_REF] Carruthers | De novo transcriptome assembly, annotation and comparison of four ecological and evolutionary model salmonid fish species[END_REF] and consolidate resources to address this challenging key task. While the number of markers cannot be considered sufficient to accurate phylogeny estimation [START_REF] Pyron | Post-molecular systematics and the future of phylogenetics[END_REF] and their nature may lead to conflicting results (e.g. [START_REF] Reddy | Why do phylogenomic data sets yield conflicting trees? Data type influences the avian tree of life more than taxon sampling[END_REF], results by [START_REF] Collins | An in silico comparison of protocols for dated phylogenomics[END_REF] suggested good performance of SNP data coming from restriction-site-associated DNA (RAD) analysis to address relatively recent and shallow phylogenies. However, relevance of models behind RAD-based and other SNP analysis remains to be considered further [START_REF] Bravo | Embracing heterogeneity: coalescing the Tree of Life and the future of phylogenomics[END_REF]. Among methods, the multispecies coalescent (MSC) emerged to tackle complex phylogenies in the age of increasing access to genomic data (e.g., [START_REF] Rannala | Efficient Bayesian species tree inference under the multispecies coalescent[END_REF]. MSC is a statistical framework based on coalescent theory that explicitly accounts for gene tree discordance due to incomplete lineage sorting and was initially devoted to identify 'species' as entities between which genetic exchanges have been negligible. It may now address more complex demographic modelling during speciation (e.g., [START_REF] Hey | Phylogeny estimation by integration over isolation with migration models[END_REF][START_REF] Long | The effect of gene flow on coalescent-based species-tree inference[END_REF], have been extended to phylogenetic network as the multispecies network coalescent (e.g., [START_REF] Wen | Bayesian inference of reticulate phylogenies under the multispecies network coalescent[END_REF][START_REF] Zhang | Bayesian inference of species networks from multilocus sequence data[END_REF], and recognized as "broadly" efficient [START_REF] Hillis | Species delimitation in herpetology[END_REF][START_REF] Kapli | Phylogenetic tree building in the genomic age[END_REF][START_REF] Rannala | Efficient Bayesian species tree inference under the multispecies coalescent[END_REF].

Guidelines and tools regarding coalescent-based species delimitations and further testing procedures are regularly enriched (e.g., [START_REF] Hey | Population genetics and objectivity in species diagnosis[END_REF][START_REF] Carstens | How to fail at species delimitation[END_REF][START_REF] Flouri | A Bayesian implementation of the multispecies coalescent model with introgression for phylogenomic analysis[END_REF][START_REF] Jones | Algorithmic improvements to species delimitation and phylogeny estimation under the multispecies coalescent[END_REF][START_REF] Kapli | Phylogenetic tree building in the genomic age[END_REF][START_REF] Liu | Modern phylogenomics: building phylogenetic trees using the multispecies coalescent model[END_REF][START_REF] Luo | Comparison of methods for molecular species delimitation across a range of speciation scenarios[END_REF]. In fish, ancestral geographic distribution or estimation of divergence times among closely related lineages benefited of such new statistical modelling approaches in sticklebacks [START_REF] Fang | Estimating uncertainty in divergence times among three-spined stickleback clades using the multispecies coalescent[END_REF]. In cichlids, [START_REF] Olave | Implementing large genomic single nucleotide polymorphism data sets in phylogenetic network reconstructions: a case study of particularly rapid radiations of cichlid fish[END_REF] illustrated capacities to deal with recent radiation events that might also be present in some lacustrine trout (e.g., S. ischchan in Lake Sevan, Armenia, in which four sympatric morphs were recorded; [START_REF] Dadikyan | Fishes of Armenia[END_REF].

Despite access to statistical procedures and genomic resources, species are hypotheses and not inherently subjectives, because of artificial cut-offs in the process of divergence (e.g. [START_REF] Hey | The mind of the species problem[END_REF][START_REF] De Queiroz | Species concepts and species delimitation[END_REF][START_REF] Pante | Species are hypotheses: avoid connectivity assessments based on pillars of sand[END_REF]. MSC -or any other method (e.g. approximate Bayesian computations) -is then not a magic bullet and improving trout phylogeny that describe a speciation process by analyzing genome-wide data sets is not sufficient to establish criteria for taxon delimitation (e.g. [START_REF] Chambers | The multispecies coalescent over-splits species in the case of geographically widespread taxa[END_REF][START_REF] Stanton | More grist for the mill? Species delimitation in the genomic era and its implications for conservation[END_REF][START_REF] Sukumaran | Multispecies coalescent delimits structure, not species[END_REF][START_REF] Willis | no. Or, arbitrary assignment of lineages to species obscures the diversification processes of Neotropical fishes[END_REF]. Result accuracy and criteria for taxon delimitation have to be considered in light of complementary knowledge coming from different perspectives, including phenotypic and ecological information [START_REF] Barley | Impact of model violations on the inference of species boundaries under the multispecies coalescent[END_REF][START_REF] Campillo | Model-based species delimitation: are coalescent species reproductively isolated?[END_REF][START_REF] Hillis | Species delimitation in herpetology[END_REF][START_REF] Solís-Lemus | Bayesian species delimitation combining multiple genes and traits in a unified framework[END_REF][START_REF] Sukumaran | Multispecies coalescent delimits structure, not species[END_REF], as well as geographical/distributional consideration (e.g., [START_REF] De Queiroz | Species concepts and species delimitation[END_REF][START_REF] Hillis | Species delimitation in herpetology[END_REF][START_REF] Hillis | The detection and naming of geographic variation within species[END_REF]. In the roundtail chub (G. robusta) complex, [START_REF] Copus | Geopolitical species revisited: genomic and morphological data indicate that the roundtail chub Gila robusta species complex (Teleostei, Cyprinidae) is a single species[END_REF] showed that morphological variation reflected phenotypic plasticity and that only molecular variation could offer consistent material for taxa delimitation. Willis (2017) also discussed the real necessity of species delimitation in peacock cichlids (Cichla pinima species complex), because their evolutionary history could not be unambiguously rendered as discrete units. Concurrently, [START_REF] Kautt | Lessons from a natural experiment: Allopatric morphological divergence and sympatric diversification in the Midas cichlid species complex are largely influenced by ecology in a deterministic way[END_REF] showed that Midas cichlid species complex from Nicaraguan crater's lakes can be described as independent evolutionary lineages that established differences in morphology, ecological settings and distribution, suggesting the likely existence of criteria for taxa/species delimitation in less than 2000 generations.

'Going large' offers to explore many facets of trout speciation, strengthen the interplay between systematics, phylogeography and speciation genomics [START_REF] Galtier | Delineating species in the speciation continuum: A proposal[END_REF], and operate to renew links with conservation issues [START_REF] Coates | Genetic diversity and conservation units: dealing with the speciespopulation continuum in the age of genomics[END_REF][START_REF] Stanton | More grist for the mill? Species delimitation in the genomic era and its implications for conservation[END_REF][START_REF] Raposo | On the notions of taxonomic 'impediment', 'gap', 'inflation' and 'anarchy', and their effects on the field of conservation[END_REF]. It may allow for the definition of shared operational criteria for trout (sub-)species delimitations and to set aside a current situation where many trout taxa -while reproductively non-isolated -have been described as tips of one unknown phylogeny. [START_REF] Carstens | How to fail at species delimitation[END_REF] and others proposed to be conservative when delimiting species and the current situation militates for recognizing only few trout species. Actually only S. ohridanus and S. obtusirostris seem to have receive (relatively) sufficient molecular support as independent taxa. Much work has still to be done and trout from the Anatolian and Taurus regions are certainly the most interesting taxa that might contribute to elucidate the trout phylogenetic puzzle.

Finally, because of the availability of OTUs present only as museum specimens (e.g. S. pallaryi), the study of ancient or degraded DNA has also to be more deeply considered in trout. Few studies considered museum specimens, only using mtDNA [START_REF] Splendiani | The effects of paleoclimatic events on Mediterranean trout: Evidences from ancient DNA[END_REF][START_REF] Splendiani | Museum samples could help to reconstruct the original distribution of Salmo trutta complex in Italy[END_REF][START_REF] Levin | High-throughput sequencing of the mitochondrial genomes from archived fish scales: an example of the endangered putative species flock of Sevan trout Salmo ischchan[END_REF][START_REF] Tougard | Salmo macrostigma (Teleostei, Salmonidae): Nothing more than a brown trout (S. trutta) lineage?[END_REF]. Study of nuclear ancient DNA is challenging, but may improve knowledge [START_REF] Chassaing | Phylogeography of the European sturgeon (Acipenser sturio): A critically endangered species[END_REF][START_REF] Lopez | Genomics of natural history collections for understanding evolution in the wild[END_REF].

In the meantime

If the bar is too high before to gather extensive genome-wide data, a pragmatic approach remains the study of complete mitogenomes. Trout mitogenomes are recent and now retrieved from both contemporary and archived samples (e.g., [START_REF] Nedoluzhko | Two complete mitochondrial genomes of extinct form of the Sevan trout Salmo ischchan danilewskii[END_REF][START_REF] Sahoo | The complete mitogenome of brown trout (Salmo trutta fario) and its phylogeny[END_REF][START_REF] Tougard | Salmo macrostigma (Teleostei, Salmonidae): Nothing more than a brown trout (S. trutta) lineage?[END_REF] first S. salar mitogenome by [START_REF] Hurst | The complete mitochondrial DNA sequence of the Atlantic salmon, Salmo salar[END_REF].

While mtDNA is uniparentally inherited, produces over-pronounced geographic structuring and is a notable source of mito-nuclear discordance (i.e. produces very partial pictures of connectivity and gene flow among OTUs; [START_REF] Petit | Gene flow and species delimitation[END_REF][START_REF] Toews | The biogeography of mitochondrial and nuclear discordance in animals[END_REF], phylogenies using complete mitogenome remain largely indicative of the speciation process and paleogeographic events [START_REF] Ben Chehida | Mitochondrial genomics reveals the evolutionary history of the porpoises (Phocoenidae) across the speciation continuum[END_REF][START_REF] Jacobsen | Mitogenome sequencing reveals shallow evolutionary histories and recent divergence time between morphologically and ecologically distinct European whitefish (Coregonus spp.)[END_REF][START_REF] Wang | Complete mitochondrial genomes of eight seahorses and pipefishes (Syngnathiformes: Syngnathidae): insight into the adaptive radiation of syngnathid fishes[END_REF]; see also [START_REF] Collins | An in silico comparison of protocols for dated phylogenomics[END_REF]). Mitogenomes enabled support to complex situations in which specific status was not previously assessed and/or ambiguous (e.g. [START_REF] Beheregaray | Genome-wide data delimits multiple climate-determined species ranges in a widespread Australian fish, the golden perch (Macquaria ambigua)[END_REF][START_REF] Buckley | Phylogenomic history of enigmatic pygmy perches: implications for biogeography, taxonomy and conservation[END_REF], providing also support for a more integrative and corroborative taxonomy in which morphology and molecular species delineation have been shown to match [START_REF] Pedraza-Marrón | Genomics overrules mitochondrial DNA, siding with morphology on a controversial case of species delimitation[END_REF] or not [START_REF] Vernygora | Delimitation of Alosa species (Teleostei: Clupeiformes) from the Sea of Azov: integrating morphological and molecular approaches[END_REF].

In the meantime, we may also ask ourselves if progress in molecular trout studies do not also depend on a large cooperative research network that may gather samples from many OTUs, from many selected locations (and museums) and further ecological information to push trout research to new areas. If local research groups exist (e.g., Balkan

Trout Restoration Group: http://www.balkan-trout.com/research.htm), transnational studies remain few, mostly binational, and large-scale international programs regarding trout date back to two decades ago (TroutConcert;Laikre, 1999). New collaborations are certainly necessary to reach novel achievements and make the mirage of trout molecular studies disappear to both splitters and lumpers.

Captions of the Figures

Fig. 1: Evolution of the number of publications over the 1989-2020 period dealing with trout systematics found in public reference databases (Web of Sciences, Google Scholar, PubMed) in June 2020 using "Salmo", "phylogeny", or "systematics" as queries, then curated (e.g. reviews reporting data from former studies were discarded to avoid to count a study twice). A reference was considered relevant when, at least, two Salmo taxonomic species or S. trutta evolutionary lineages were included in the studied dataset, and phylogenetic relationships were represented by a tree or a network. The full list of references (N = 128) used in this study is available upon request to the contact author. MtDNA: PCR-based sequence of mitochondrial DNA genes; nucDNA: PCR-based sequence of nuclear genes and microsatellites; NGS: data obtained through next-generation sequencing technologies, including nuclear SNPs and mitogenomes. Studies strictly based on allozymic data were not considered. (1140 base pairs) genes. Numbers at nodes are for posterior probabilities (≥0.80) and bootstrap percentages (≥50%), while "-" are for nodes weakly supported in either the Bayesian or maximum likelihood analyses. Lineage and species names are indicated on the right. Outside the Atlantic salmon taken as outgroup, note that only four taxa are considered in this tree (S. trutta, S. ohridanus, S. obtisrostris and S. marmoratus as the marble trout lineage is often considered a separate OTU [e.g., [START_REF] Pustovrh | Molecular phylogeny of Salmo of the western Balkans, based upon multiple nuclear loci[END_REF]), meaning this phylogeny includes only 17.4% (4 out of 23) species recognized by IUCN. The horizontal bar under the phylogenetic tree indicates the number of nucleotide substitutions per site. For details about sampling and phylogenetic analyses, see [START_REF] Tougard | Salmo macrostigma (Teleostei, Salmonidae): Nothing more than a brown trout (S. trutta) lineage?[END_REF].

Accepted Article Accepted Article

(

  2018) used SNPs to document the divergence in the S.trutta/S. ferox 'species' pair occurring in Loch Maree catchment (Scotland), but this did not represent a phylogenetic study. Only[START_REF] Lecaudey | Inferring phylogenetic structure, hybridization and divergence times within Salmoninae (Teleostei: Salmonidae) using RAD-sequencing[END_REF] used SNPs in a phylogenetic context, but they were interested in higher phylogenetic relationships than the ones sustaining the brown trout complex.

Fig. 2 :

 2 Fig. 2: Simplified Bayesian phylogenetic tree reconstructed from a concatenated dataset

Acknowledgements

The authors thank A. Sabatini and F. Palmas for invitation to the Associazione Italiana Ittiologi Acque Dolci thematic congress on the Biodiversity of Mediterranean Salmonids held in Cagliari, Italy, 2019, and further exchange and discussions with participants that inspired this article. We thank P. Berrebi and anonymous reviewers for thoughtful comments. M. Oral was supported by a visiting grant from the MUSE 'Explore' programme, publicly funded through ANR (French National Research Agency) under the "Investissements d'avenir" program (ANR-16-IDEX-0006), and the Erasmus+ program of the European Union.