domain: sdv sde

text:
Allan Debelle
email: allandebelle@gmail.com

Alexandre Courtiol

Michael G Ritchie

Rhonda R Snook

Mate choice intensifies motor signalling in Drosophila

Keywords: courtship song, Drosophila pseudoobscura, experimental evolution, interpulse interval, mate choice, motor performance, sexual selection

Mate choice has the potential to act on the evolution of motor performance via its direct influence on motor sexual signals. However, studies demonstrating this are rare. Here, we perform an in-depth analysis of Drosophila pseudoobscura courtship song rate, a motor signal under mate choice in this species, and analyse the response of this signal to sexual selection manipulation using experimental evolution. We show that manipulating the opportunity for sexual selection led to changes in song production rate and singing endurance, with males from the polyandrous populations producing faster song rates over longer time periods than males from monogamous populations. We also show that song rate is correlated with estimates of overall courtship vigour. Our results suggest that the action of mate choice on a motor signal has affected male motor performance displayed during courtship. We consider potential selective benefits associated with changes in motor performance, including condition-dependent signalling, and discuss the implications of these results for the study of motor signals under sexual selection.

Motor performance, or vigour [START_REF] Darwin | The Origin of Species by Means of Natural Selection Or the Preservation of Favoured Races in the Strugggle for Life[END_REF][START_REF] Darwin | The Descent of Man, and Selection in Relation to Sex[END_REF], is the ability of an individual to repeatedly perform energetically-costly motor acts [START_REF] Byers | Female mate choice based upon male motor performance[END_REF]. As this ability often has drastic fitness consequences (e.g. determining the ability to escape predators, forage or capture preys), its evolution is often driven by natural selection [START_REF] Byers | Female mate choice based upon male motor performance[END_REF][START_REF] Irschick | Integrating function and ecology in studies of adaptation: investigations of locomotor capacity as a model system[END_REF]). Yet, sexual selection also has the potential to affect the evolution of motor performance, when mate choice or mate competition targets motor signals (i.e. signals involving any kind of sustained muscular activity such as threat displays, courtship displays such as dances, or acoustic and vibratory signals; [START_REF] Bonduriansky | Sexual selection and conflict as engines of ecological diversification[END_REF][START_REF] Husak | Sexual selection on locomotor performance[END_REF]. Because such signals typically require high-speed muscle contractions that are energetically-costly to produce [START_REF] Lailvaux | A functional perspective on sexual selection: insights and future prospects[END_REF], they have the potential to be reliable indicators of a signaller's overall motor capacities, and thus of the individual's current condition [START_REF] Byers | Female mate choice based upon male motor performance[END_REF][START_REF] Clark | The role of power versus energy in courtship: what is the "energetic cost" of a courtship display?[END_REF][START_REF] Lailvaux | A functional perspective on sexual selection: insights and future prospects[END_REF][START_REF] Oufiero | Evaluating performance costs of sexually selected traits[END_REF]. Hence, by directly influencing the evolution of a given motor signal, sexual selection may lead to a correlated increase of the overall motor capacities of signallers [START_REF] Byers | Female mate choice based upon male motor performance[END_REF][START_REF] Clark | The role of power versus energy in courtship: what is the "energetic cost" of a courtship display?[END_REF][START_REF] Mowles | Repetitive signals and mate choice: insights from contest theory[END_REF][START_REF] Ryan | Energy, calling, and selection[END_REF].

Although potential links between motor sexual signals and motor performance have received significant attention in the recent literature [START_REF] Byers | Female mate choice based upon male motor performance[END_REF][START_REF] Irschick | How does selection operate on whole-organism functional performance capacities? A review and synthesis[END_REF][START_REF] Mowles | Repetitive signals and mate choice: insights from contest theory[END_REF], their investigation has so far been restricted to two issues; the link between motor signals involved in mate competition and overall motor performance [START_REF] Andersson | Sexual selection[END_REF][START_REF] Byers | Female mate choice based upon male motor performance[END_REF][START_REF] Lailvaux | A functional perspective on sexual selection: insights and future prospects[END_REF] and the link between motor signals involved in mate choice and non-motor measures of mate condition (e.g. offspring production, growth rate, etc.; [START_REF] Irschick | How does selection operate on whole-organism functional performance capacities? A review and synthesis[END_REF]. For example, a link between male dominance display and running endurance has been shown in Anolis lizards [START_REF] Perry | Locomotor performance and social dominance in male Anolis cristatellus[END_REF], and a correlation between male song structure and offspring survival was found in the zebra finch Taeniopygia guttata [START_REF] Woodgate | Male song structure predicts reproductive success in a wild zebra finch population[END_REF]). Yet, mate choice for motor signals may also affect the evolution of overall mate motor performance [START_REF] Byers | Female mate choice based upon male motor performance[END_REF][START_REF] Clark | The role of power versus energy in courtship: what is the "energetic cost" of a courtship display?[END_REF][START_REF] Mowles | Repetitive signals and mate choice: insights from contest theory[END_REF][START_REF] Ryan | Energy, calling, and selection[END_REF][START_REF] Ryan | Directional patterns of female mate choice and the role of sensory biases[END_REF]. Numerous studies have shown that mate choice could drive the evolution of motor signals, but evidence for a correlated effect on overall motor performance is still lacking [START_REF] Byers | Female mate choice based upon male motor performance[END_REF][START_REF] Fusani | Physiological control of elaborate male courtship: female choice for neuromuscular systems[END_REF][START_REF] Mowles | Repetitive signals and mate choice: insights from contest theory[END_REF].

A suitable approach to investigate this question is to determine how mate choice affects a motor signal over evolutionary time, and then examine whether these changes also results in changes in aspects of overall motor performance. As a widely studied acoustic mating signal, the pulse production rate of Drosophila male courtship song is a prime candidate for such a study, for multiple reasons. First, Drosophila courtship song consists of a series of repeated pulses created by rapid wing vibrations, obtained via high-speed contractions of thoracic muscles [START_REF] Ewing | The neuromuscular basis of courtship song in Drosophila: the role of the direct and axillary wing muscles[END_REF][START_REF] Ewing | The neuromuscular basis of courtship song in Drosophila: The role of the indirect flight muscles[END_REF][START_REF] Shirangi | Motor control of Drosophila courtship song[END_REF]. The rate at which these pulses are produced (commonly reported as the interpulse interval, or IPI, representing the inverse of pulse rate) is thus likely to be a physically challenging motor trait. Next, the song pulse rate is a key target of female choice in several Drosophila species. It is involved in the sexual isolation between D. melanogaster and D. simulans, and in intraspecific mate choice in D. melanogaster, D. montana and D. pseudoobscura [START_REF] Bennet-Clark | Pulse interval as a critical parameter in the courtship song of Drosophila melanogaster[END_REF][START_REF] Debelle | Evolution of divergent female mating preference in response to experimental sexual selection[END_REF][START_REF] Kyriacou | The function of courtship song rhythms in Drosophila[END_REF][START_REF] Ritchie | Drosophila song as a species-specific mating signal and the behavioural importance of Kyriacou & Hall cycles in D. melanogaster song[END_REF][START_REF] Veltsos | Sexual selection on song and cuticular hydrocarbons in two distinct populations of Drosophila montana[END_REF][START_REF] Williams | Courtship songs of Drosophila pseudoobscura and D. persimilis. II. Genetics of species differences[END_REF].

Then, the fact that song pulse rate is a target of female choice has been further demonstrated by showing the coevolution of pulse rate and female preference for pulse rate in experimental populations of D. pseudoobscura [START_REF] Debelle | Evolution of divergent female mating preference in response to experimental sexual selection[END_REF]. Finally, a direct action of male-male competition on pulse rate evolution is improbable. Courtship song is a near-field acoustic signal produced within 2.5-5mm of the female's head [START_REF] Bennet-Clark | Acoustics of insect song[END_REF], 1998), rendering its accurate reception by surrounding male competitors unlikely [START_REF] Morley | Directional cues in Drosophila melanogaster audition: structure of acoustic flow and inter-antennal velocity differences[END_REF]. Hence, and although playing artificial courtship songs to males in playback experiments triggers male locomotion [START_REF] Eberl | The effect of male competition on the courtship song of Drosophila melanogaster[END_REF][START_REF] Von Schilcher | The role of auditory stimuli in the courtship of Drosophila melanogaster[END_REF], varying pulse rate does not have an effect on male courtship behaviour [START_REF] Talyn | The role of courtship song in sexual selection and species recognition by female Drosophila melanogaster[END_REF].

Therefore, Drosophila song pulse rate has all the necessary characteristics to be a suitable candidate for this study.

Yet, how song pulse rate relates to the evolution of male motor performance has so far not been investigated. This may be because the rate of Drosophila courtship song is commonly considered to be static, i.e. stable in time and independent of male motor capacities. Like many other acoustic signals, pulse rate is thus usually measured at a single time point or averaged over the entire courtship sequence [START_REF] Tauber | Acoustic communication in Drosophila[END_REF]; but see a notable exception in [START_REF] Arthur | Multi-channel acoustic recording and automated analysis of Drosophila courtship songs[END_REF]. That restricted view of this motor signal makes it impossible to know how much this trait depends on a male's motor capacities [START_REF] Irschick | Integrating function and ecology in studies of adaptation: investigations of locomotor capacity as a model system[END_REF]. Another important aspect in studying the action of mate choice on motor performance evolution is to measure how the trait under mate choice is associated with other traits. By targeting pulse rate, mate choice could lead to a correlated response on other motor traits [START_REF] Gerhardt | Experimental analysis of multivariate female choice in gray treefrogs (Hyla versicolor): evidence for directional and stabilizing selection[END_REF][START_REF] Lande | The measurement of selection on correlated characters[END_REF], and therefore investigating these associations is essential to understand how mate choice may influence the evolution of motor performance beyond pulse rate.

In this study, we examine closely the production of a motor signal involved in mate choice, and quantify how manipulating the opportunity for sexual selection [START_REF] Jones | On the opportunity for sexual selection, the Bateman gradient and the maximum intensity of sexual selection[END_REF] influences the evolution of motor performance that is displayed during courtship. For that purpose, we study the production of D. pseudoobscura pulse rate over the duration of courtship, and explore its response to a long-term experimental manipulation of the opportunity for sexual selection in D. pseudoobscura populations (>100 generations of experimental evolution of elevated polyandry or enforced monogamy). An analysis of these experimental lines performed after 30 generations of selection has found that mean pulse rate had responded to sexual selection manipulation, and had become faster in males from polyandrous lines compared to monogamous lines [START_REF] Snook | Experimental manipulation of sexual selection and the evolution of courtship song in Drosophila pseudoobscura[END_REF]. This previous study was however performed on a restricted number of individuals and limited to the examination of average pulse rate. As a consequence, this study did not allow investigating potential differences in pulse rate production over time, which is necessary to study overall motor performance. Here, we perform an in-depth study of pulse rate production over time in our experimental lines after much longer evolution, and analyse the effect of sexual selection manipulation on motor signalling.

Our main prediction is that an increased opportunity for sexual selection will lead to the evolution of more intense male signalling characteristics and thus to an increased motor performance. For this end, we look at the detailed structure of pulse rate production over courtship time, to uncover potential sources of motor performance difference between males.

We then compare pulse rate production between the sexual selection treatments, to study whether pulse rate production responded to sexual selection manipulation. Finally, we examine associations between pulse rate and other motor courtship traits, to test whether pulse rate may be correlated with overall courtship vigour.

METHODS

The courtship behaviour of D. pseudoobscura has been described in detail elsewhere [START_REF] Brown | Courtship behavior in the Drosophila obscura group. I: D. pseudoobscura[END_REF][START_REF] Ewing | The courtship songs of Drosophila[END_REF]). Courtship song is produced by the vibration of one or both male wing(s), and consists of two main components: a low-repetition rate song (LRR) and a high-repetition rate song (HRR) (Fig. 1). LRR consists of high-amplitude polycyclic pulses and is generally produced first, while the male orients in the direction of the female and approaches her, by flicking one or both wing(s) in a scissoring movement. Once the male has reached the female, he extends the wing that is nearest the female's head to 90° and vibrates it rapidly, producing a burst of HRR, characterised by a high number of low-amplitude polycyclic pulses and an increase in pulse rate (i.e. a shorter duration between two consecutive pulses in a burst of song, and thus a shorter interpulse interval). The male will then generally attempt to mount the female and copulate. If the female refuses to mate, the male will start another courtship sequence, including another round of song bursts. As HRR pulse rate is the main target of female preference in this species [START_REF] Debelle | Evolution of divergent female mating preference in response to experimental sexual selection[END_REF][START_REF] Snook | Experimental manipulation of sexual selection and the evolution of courtship song in Drosophila pseudoobscura[END_REF][START_REF] Williams | Courtship songs of Drosophila pseudoobscura and D. persimilis. II. Genetics of species differences[END_REF], we focus on HRR song in this study (but provide a similar analysis of LRR song in Appendix A).

Sexual Selection Treatments

An ancestral wild-caught population of D. pseudoobscura, a naturally polyandrous species (more than 80% of wild-caught females have been shown to be inseminated by up to two males at any given time; [START_REF] Cobbs | Multiple insemination and male sexual selection in natural population of Drosophila pseudoobscura[END_REF], was used to create the selection lines. The establishment and maintenance of the selection lines are described in detail elsewhere [START_REF] Crudgington | Experimental removal and elevation of sexual selection: does sexual selection generate manipulative males and resistant females[END_REF]. In brief, from an ancestral population derived from Tucson (Arizona, US), 4 replicates (replicate 1, 2, 3 and 4) of two sexual selection treatments were initiated. To modify the opportunity for sexual selection at each generation, adult sex-ratio in vials is manipulated by either confining one female with a single male ('monogamy' treatment; M) or one female with 6 males ('elevated polyandry' treatment; E) in vials. Both intra-and inter-sexual selection are relaxed in the monogamy treatment whereas both types of sexual selection are increased in the polyandry treatment.

As previously reported, effective population size was successfully equalized between the treatments (Ne>100 for all the populations; [START_REF] Snook | A test and review of the role of effective population size on experimental sexual selection patterns[END_REF]. At each generation and in each population independently, offspring are collected and then pooled together. A random sample of this pool is used to establish the next generation using the appropriate sex-ratios. This protocol thus proportionally reflects the relative offspring production across all families. Standard vials (2.5mm x 80mm) are used to maintain the selection lines, with a 28-day generation time. Bottles (57 mm x 132 mm) are used to maintain the ancestral population, with an equal sex-ratio of adult flies. Therefore, a total of 8 selection lines (M1, M2, M3, M4 and E1, E2, E3, E4) and one ancestral population are maintained and kept at 22 o C on a 12L:12D cycle, using standard food media and added live yeast.

Experimental Flies

The flies used in this experiment were from the following generations: 111 and 112 for E1 and M1, 110 and 111 for E2 and M2, 109 and 110 for E3 and M3, 107 and 108 for E4 and M4. To generate the experimental flies, 50 reproductively mature adults of each selection line (25 males and 25 females) were used as parents and kept in mass-cultures, providing a common mating set up for the parents of both sexual selection treatments. The resulting larvae were raised in controlled density vials (100 first instar larvae per food vial), to standardize the larval rearing environment and relax selection. The flies were collected and sexed on the day of emergence, using CO 2 anaesthetisation. Males from each population were kept in yeasted food vials of 10 individuals from the day of emergence to day 4, and then transferred to individual yeasted food vials the day before the recording. We used ancestral females for male courtship song recording to standardise female response. Ancestral females were collected and kept in vials of 10 individuals until used for the song recording experiment.

Ancestral females were mated to ancestral males the day before the experiment to reduce their receptivity and prevent them from mating with the focal recorded male within the 5 minutes of the trial. Female receptivity is drastically reduced in the 24 hours following a mating, and thus the probability of remating for the ancestral females used in this experiment would be nearly zero [START_REF] Crudgington | Experimental removal and elevation of sexual selection: does sexual selection generate manipulative males and resistant females[END_REF][START_REF] Snook | The risk of sperm competition and the evolution of sperm heteromorphism[END_REF]. This method forces males to continuously court females, therefore facilitating detailed study of song production over time.

All males and females used in this experiment were 5 days old and thus reproductively mature [START_REF] Snook | Mating system evolution in sperm-heteromorphic Drosophila[END_REF]. Henceforth, reference to polyandrous or monogamous does not mean current mating situation in any experiments, but refers to the experimental sexual selection treatment from which flies are derived.

Courtship Song Recording

Recordings were performed during the flies' morning photoperiod [START_REF] Noor | Diurnal activity patterns of Drosophila subobscura and D. pseudoobscura in sympatric populations[END_REF]. Courtship song was recorded by confining one virgin selection line male with a mated ancestral female for 5 minutes in a transparent chamber (15 mm x 4 mm) in an Insectavox [START_REF] Gorczyca | The INSECTAVOX, an integrated device for recording and amplifying courtship songs of Drosophila[END_REF]. Recordings took place over the course of 12 days. All 8 lines were randomised across and within days of recording. Each male was only recorded once, and 60 males were recorded per selection line. The Insectavox was connected to a Toshiba Satellite Pro S300-117 laptop, and sound was recorded using Audacity (v. 1.3.11). All songs were digitised after filtering with a Fern EF5-04 filter, band-passed between 100 and 800 Hz. After the experiments, recordings were manually prepared for software analysis by silencing parts of the recording without song using Audacity (v. 1.3.11). Recordings were then analysed using a custom script from the software DataView [START_REF] Heitler | DataView: a tutorial tool for data analysis. template-based spike sorting and frequency analysis[END_REF], allowing the detection of the position of each 'song event' (pulses and bursts) in a recording. Intrapulse frequency for both LRR and HRR songs was obtained using a fast Fourier Transform (FFT) in DataView (FFT duration=16ms, FFT window=hamming, percentage overlap=50%).

Temperature and Body Size

To understand more extensively how pulse rate is related to male motor capacities, it is informative to examine how it covaries with two key bioenergetic factors that can affect acoustic communication in insects: temperature and body size [START_REF] Bailey | Acoustic Behaviour of Insects. An Evolutionary Perspective[END_REF][START_REF] Bennett | Thermal dependence of locomotor[END_REF][START_REF] Gillooly | The energetic basis of acoustic communication[END_REF]. Temperature -which strongly influences muscle contraction rate -is tightly associated with motor power and endurance in ectotherms via its effect on metabolic rate [START_REF] Gillooly | Effects of size and temperature on metabolic rate[END_REF]), and has a major impact on the temporal components of acoustic signals in insects [START_REF] Bailey | Acoustic Behaviour of Insects. An Evolutionary Perspective[END_REF], including Drosophila courtship song traits [START_REF] Noor | Courtship songs of Drosophila pseudoobscura and D. persimilis: analysis of variation[END_REF][START_REF] Ritchie | Genetic variability of courtship song in a population of Drosophila melanogaster[END_REF][START_REF] Ritchie | Rapid evolution of courtship song pattern in Drosophila willistoni sibling species[END_REF][START_REF] Ritchie | Characterization of female preference functions for Drosophila montana courtship song and a test of the temperature coupling hypothesis[END_REF]. Likewise, body size -a target of sexual selection -is positively correlated with motor performance, notably due to the increased power provided by larger muscles [START_REF] Biewener | Animal locomotion[END_REF][START_REF] Carrier | Ontogenetic limits on locomotor performance[END_REF]. Thus, including these variables in our analyses will give a better understanding of how much pulse rate production depends on male motor capacities, and thus of how the physiological properties of Drosophila courtship song can have an impact on its evolution as a sexual signal.

As the light within the Insectavox generates inevitable random small variations in temperature, we examine in detail how song traits vary with these minor changes in temperature. Temperature was measured within the chamber every 10 seconds (+-0.01C) using a Testo 735-1 thermometer (Testo Limited, United Kingdom) and recorded for each burst of song in each recording. This temperature variation was then included as a covariate in the song analyses (temperature was either calculated for each burst in the case of HRR pulse rate, or averaged over all bursts for the other traits).

To estimate how body size could associate with pulse rate production, the size of the singing male was included in the analyses. The length of wing vein IV of each individual was measured after the experiment (wing vein length has been shown to be a good estimator of body size in Drosophila species, (e.g. [START_REF] Crudgington | Experimental removal and elevation of sexual selection: does sexual selection generate manipulative males and resistant females[END_REF][START_REF] Gilchrist | Rapid evolution of wing size clines in Drosophila subobscura[END_REF][START_REF] Robertson | Heterozygosity, environmental variation and heterosis[END_REF][START_REF] Sokoloff | Morphological Variation in Natural and Experimental Populations of Drosophila pseudoobscura and Drosophila persimilis[END_REF]. Wings were mounted in a 30% glycerol-70% ethanol medium, images taken using a Motic camera and Motic Images Plus 2.0 software (Motic Asia, Hong Kong) and then measured with ImageJ (v. 1.44e;[START_REF] Abramoff | Image processing with ImageJ[END_REF].

Courtship Traits Analysis

The different courtship traits analysed in this study are represented in Fig. 1. All the statistical analyses were performed in R (v. 3.3.2;R Development Core Team, 2005).

We first tested for differences between the sexual selection treatments in body size and in their probability of producing song. As HRR interpulse interval (i.e. the inverse of pulse rate) is not constant over time but lengthens as courtship time increases (see Fig. B1 in Appendix B), we then conducted a detailed analysis of pulse rate production over courtship time, and compared pulse rate production between treatments. Finally, we performed multivariate analyses on all courtship traits to study phenotypic correlations between pulse rate and other courtship traits, and to test whether sexual selection manipulation modified these associations.

Differences In Body Size and Singing Probability Between The Sexual Selection Treatments:

Potential differences in body size between the sexual selection treatments were analysed using a univariate linear mixed model (LMM), in which the sexual selection treatment of the recorded male (E or M) was included as a fixed effect, and the male replicate population (M1, M2, M3, M4, E1, E2, E3 or E4) included as a Gaussian random effect nested within sexual selection treatment. The model was fitted using maximum likelihood estimation, with a Gaussian error distribution. The difference in the probability of singing (i.e. the probability of a male producing at least one burst of HRR during the 5-min recording) between the sexual selection treatments was analysed using the same model structure but fitted a generalized linear mixed model (GLMM) for the binomial family. Both models were fitted using the package spaMM [START_REF] Rousset | Testing environmental and genetic effects in the presence of spatial autocorrelation[END_REF] and estimates were compared to zero using parametric bootstraps which were consistent with results from model comparison using asymptotic likelihood ratio tests.

Detailed Analysis Of Pulse Rate Production Over Time:

To distinguish between HRR interpulse and interburst interval (i.e. the interval of time between the last pulse of a burst and the first pulse of the following burst), an upper threshold was determined visually by plotting the distribution of the duration between two pulses (threshold = 55 ms; the average HRR interpulse interval is approximately 38 ms in D. pseudoobscura, [START_REF] Noor | Courtship songs of Drosophila pseudoobscura and D. persimilis: analysis of variation[END_REF][START_REF] Snook | Experimental manipulation of sexual selection and the evolution of courtship song in Drosophila pseudoobscura[END_REF]. To allow sufficient HRR interpulse interval values for each burst, we only included recordings with at least 10 interpulse interval values (i.e. the overall average number of HRR pulses per burst for both E and M males is 17; see Figure B2 for more details).

Variation in individual interpulse interval values along the courtship sequence was analysed by fitting a univariate LMM, using the function glmmPQL() of the MASS package [START_REF] Venables | Modern applied statistics with S (Fourth)[END_REF]. This enables correcting for temporal autocorrelation between consecutive interpulse interval values within a burst. We thus included in the model a fourth-order autoregressive moving-average (corARMA) function for autocorrelation, using the pulse position in a burst (i.e. 1, 2, 3, etc.) as a time covariate, and the burst identity (1735 levels) nested within replicate (8 levels) as a grouping factor (nlme package; Pinheiro, Bates, DebRoy, Sarkar, & R Core Team, 2016). We also included two covariates indicating the position of the interpulse interval value within the courtship sequence, the burst position in the recording (i.e. 1, 2, 3, etc.) and the pulse position within a burst (i.e. 1, 2, 3, etc.), to test for a lengthening of interpulse interval over courtship duration (both within bursts and along bursts; see Fig. 1 for more details). The interactions between sexual selection treatment and the two event position covariates (burst position and pulse position) were included in the model, as well as their three-way interaction. This allowed us to assess how interpulse interval variation changes depending on the quantity of song already produced, and also to test whether interpulse interval variation over time is consistent between the two treatments. The interaction between temperature and burst position was also added, to test for an effect of temperature on interpulse interval lengthening over time (the interaction between temperature and pulse position in the burst could not be included in the model, as the mean duration of a burst, <3s, did not allow enough time for the recorded temperature to vary). The significance of the different fixed effects was extracted from the summary table of the glmmPQL fit, which provides the t-test results comparing estimates to zero. The same pulse rate production model was also fitted while including individual body size as an additional covariate (see

Table D1 in Appendix D).
multivariate analyses require the different dependent variables to present the same number of observations, we performed the multivariate analysis using only the mean interpulse interval value of the first HRR burst produced (E and M males produced on average 14 bursts of HRR song in a recording; see Figure B2 for more details). Performing such averaging also precludes the need to consider the temporal autocorrelation that exists between successive pulses. To analyse whether interpulse interval and the other courtship traits jointly responded to sexual selection manipulation, we fitted a multivariate LMM on song data. In a multivariate LMM, the different response variables are transformed into a single univariate response variable by creating a vector that considers all observations across the different response variables sequentially [START_REF] Christensen | Advanced Linear Modeling : Multivariate, Time Series, and Spatial Data ; Nonparametric Regression and Response Surface Maximization[END_REF]. A fixed effect factor is then used to indicate the correspondence between these observations and the original response variables. We assessed the fixed effects of the mean temperature during a recording and sexual selection treatment on five courtship traits: the mean interpulse interval of the first burst of song, the mean amplitude, the mean intrapulse frequency, the total number of bursts produced, and the singing latency (i.e. the time it took a male to produce its first burst of song). All response variables were log-transformed for normalization and then converted to z-scores, to facilitate model convergence. The estimates we provide in the tables of this study correspond to the direct output from the model fit. In the text, we untransformed the estimates back to the original scale of the response variable. To do this, we calculated the exponential of the sum of (1) the product of the standard deviation of the log of the original variable by the corresponding estimate (2) the log of the mean of the original variable.

The model was fitted using the MCMCglmm package [START_REF] Hadfield | MCMC Methods for Multi-Response Generalized Linear Mixed Models: The MCMCglmm R Package[END_REF]. We ran MCMC chains for 100,000 iterations (burn-in phase), followed by five million iterations during which parameter estimates were sampled every 5000 iterations. This sampling scheme resulted in 1000 recorded estimate values for each parameter and for each model. This was sufficient to ensure that the autocorrelation between successive estimates was always lower than ±0.07.

All tests on estimates or quantities derived from estimates (e.g. correlations, see below) for this model are based on the analysis of the distribution of the 1000 records associated with a given parameter. Details about the specification of the prior distributions are given in Appendix C.

We allowed for the effects of the different covariates to differ between courtship traits. The number of estimated fixed-effect parameters was thus 15 ([1+2]x5). We estimated the variances and covariances between the response courtship traits using random effects. We computed these covariance matrices for each selection treatment (i.e. [5 variances + 10 different covariances]x2 = 30 (co)variance). We also estimated the variance between replicates separately for each courtship trait (i.e. 5 variances) considering the identity of the replicate as a random effect. We assumed the covariance between model residuals to be null, as no dependence between observations is expected with the random structure considered.

The significance of the different fixed effects was extracted from the summary table of the MCMCglmm fit. Here, the p-value is computed as twice the minimum between the probabilities that estimates sampled along the MCMC chains are either greater or lower than zero. The same model was also run while including individual body size as an additional covariate (see Table D2 in Appendix D), with 20 estimated fixed-effect parameters

([1+1+2]x5).
Estimating the variances and covariances of courtship traits allowed us to calculate the correlations between courtship traits for each treatment. Using this approach offers the advantage of estimating correlations that are not confounded by the variables included in the model as fixed (e.g. temperature) or random effects (e.g. the replicate). We then examined the significance of each individual correlation estimate, and tested for differences between the treatments, to examine whether the associations between courtship traits have changed as a result of sexual selection treatment. The significance test of these correlations was based on the analysis of estimates along the MCMC chains, as explained previously.

In all figures, the mean fixed effect estimates, hereafter referred as "predicted values" of the mixed models, are represented. Predicted values were adjusted to 22 o C, the temperature at which all populations are maintained. The 95% confidence intervals were computed for the two univariate LMMs as ±1.96 standard errors around the predicted values, with the standard error being derived from the covariance matrix of parameter estimates for fixed effects. For the GLMM, confidence intervals were computed similarly, but at the scale of the linear predictor (i.e. before the transformation from logit to probabilities). For the multivariate LMM, confidence intervals are computed as quantiles of the posterior distribution of parameter estimates along the MCMC chains. Although technically, intervals obtained this way present statistical properties that can differ from confidence intervals (e.g. Rousset, Gouy, Martinez-Almoyna, & Courtiol, 2017; they are called credibility intervals), we will refer to both types as being confidence intervals.

Predictions

First, given that energetically-costly repeated motor signals are predicted to advertise the signaller's condition [START_REF] Mowles | Repetitive signals and mate choice: insights from contest theory[END_REF], we expect pulse rate to depend on courtship effort, and thus on the quantity of song already produced by a male. For similar reasons, as motor performance should correlate positively with both temperature and body size, particularly for traits likely to act as indicators of mate condition [START_REF] Clark | The role of power versus energy in courtship: what is the "energetic cost" of a courtship display?[END_REF], we also expect pulse rate to be associated with temperature and body size. Then, if pulse rate production has been affected by sexual selection manipulation, we expect to observe faster pulse rates and a shallower slope of decline in pulse rate (i.e. a less pronounced lengthening in interpulse interval) in polyandrous males compared to monogamous males. Finally, for pulse rate to be used as an indicator of motor performance, fast pulse rates should be positively correlated with overall courtship vigour estimates (i.e. here estimated by the other motor courtship traits measured).

Ethical Note

Our design minimised the stress imposed to the individuals used in this experiment. Stress at the larval stage was prevented by controlling for larval density. At adulthood, individuals were transferred in new vials with fresh food and medium adult density. A mouth aspirator was used to gently handle live individuals throughout all the steps of the experiment. The experimental time was only 5 minutes long, after which flies were anaesthetised with CO 2 and rapidly killed in ethanol for wing measurement.

RESULTS

Differences In HRR Singing Probability Between The Sexual Selection Treatments

The probability of singing differs significantly between the sexual selection treatments (Table 1), with monogamous males having a lower probability of singing than polyandrous males (Fig. 2).

Detailed Analysis Of HRR Pulse Rate Production Over Time

This analysis, based on all bursts produced, identifies changes in interpulse interval variation between, and also within, bouts of courtship. The interpulse interval value lengthens between consecutive bursts, meaning that the rate at which males produce pulses decreases more and more as the male produces song (Table 2 and Fig. 3). Interpulse interval also lengthens within a burst, meaning that pulse rate progressively decreases during a burst too (Table 2 and Fig. 4).

Temperature: As expected, temperature strongly associates with courtship traits (Tables 2 and3). Interpulse interval is negatively associated with temperature, meaning that interpulse interval is longer at lower temperatures, as indicated by both the pulse rate production LMM and the multivariate LMM. In the latter case, interpulse interval shortens by 0.32ms (95% C.I. = -0.07 --0.57, P=0.012) when temperature increases by 1 o C (Table 3).

The pulse rate production LMM (Table 2, and Table D1 for its equivalent with body size included) also shows that the progressive shortening observed in interpulse interval is strongly correlated with temperature, with colder recording temperatures being associated with an even more pronounced lengthening in interpulse interval over courtship time (i.e. a steeper decrease in pulse rate; Fig. 5a).

Body Size: Males from polyandrous lines are larger on average than males from monogamous lines (Table 1 and Fig. 5b). When body size is included in the pulse rate production LMM (Table D1), we observe a negative effect of body size on interpulse interval.

Body size significantly influences interpulse interval both within and between bursts, meaning that larger males produce song with a shorter interpulse interval and maintained this short interpulse interval for a longer time than smaller males (Table D1 and Fig. 5c).

The multivariate LMM also reveals that, when body size is included in the model, interpulse interval shortens with increasing body size, with an increase in wing size of 1 standard deviation being associated with a reduction of 0.31ms in interpulse interval (95% C.I. = -0.03 increase in wing size of 1 standard deviation being associated with an increase in amplitude of 11.8 units (95% C.I. = 2.2 -21.4, P=0.024; Table D2).

Evolutionary Response to Sexual Selection Manipulation

The pulse rate production LMM shows a significant effect of sexual section treatment on interpulse interval, with polyandrous males producing a shorter interpulse interval (i.e. a faster pulse rate) than monogamous males (Table 2 and Fig. 3 &4). The model also shows a significant interaction between sexual selection treatment and the quantity of song already produced by a male (i.e. the burst and pulse positions in the courtship sequence), showing that the decrease in pulse rate in polyandrous males is shallower than in monogamous males.

Therefore, pulse rate differs between the sexual selection treatments, and this difference gradually widens the more males beat their wings to produce song.

Although body size is significantly different between the sexual selection treatments, including body size in the pulse rate production LMM shows a difference between the sexual selection treatments that is independent from the effect mediated by body size, with polyandrous males showing again more endurance than monogamous males (Table D1).

Analysis of the Associations Between Courtship Traits

The multivariate LMM shows that polyandrous males start to produce song earlier than monogamous males (mean difference in song latency: 5.7s; 95% C.I.= 3.5 -7.7, P=0.001;

Table 3). All other courtship traits do not show a significant difference between the sexual selection treatments (Table 3).

Table 4 presents the correlations (r) between courtship traits for each sexual selection treatment extracted from the fit of the multivariate LMM (Table 3), while Table 5 examines whether these associations differ between the sexual selection treatments (r E -r M ). The equivalent of these two tables for the multivariate LMM with body size included as a covariate are shown in the Appendix D (Table D3 andD4). Two out of the 10 correlations between courtship traits changed as a result of selection (Fig. 6 and Table 5), and both of them are associated with interpulse interval. Interpulse interval is correlated with almost all other courtship traits in the polyandry treatment (i.e. with amplitude, latency and the total number of bursts produced; Table 4), but only with amplitude in the monogamy treatment. Faster pulse rates are thus associated with shorter singing latencies, louder songs, and more bursts produced.

The multivariate LMM identified three significant associations between courtship traits that do not differ between sexual selection treatments (compare Table 4 to Table 5). In addition to the correlation between interpulse interval and amplitude, it found similar associations between the sexual selection treatments between intrapulse frequency and amplitude, and between latency and the total number of bursts produced (Table 4). The last associations found were a small positive correlation between amplitude and the total number of bursts produced, and between amplitude and latency, however these were only significant for polyandrous males, and did not significantly differ between the sexual selection treatments (Table 5).

DISCUSSION

In this study, we tested the hypothesis that mate choice influences the evolution of motor performance and predicted improved motor performance in populations subjected to more intense sexual selection. We performed a detailed analysis of the production of Drosophila pseudoobscura song pulse rate, a motor signal under mate choice in this species. We also analysed the response of this motor signal to sexual selection manipulation via experimental evolution. We showed that song pulse rate decreases with the amount of song a male has already produced, and is associated with body size and recording temperature, indicating a potential for pulse rate to act as an indicator of male condition. Consistent with this, manipulating the opportunity for sexual selection led to the evolution of faster pulse rates and improved song production endurance in males from polyandrous lines compared to males from monogamous lines. Finally, we showed that pulse rate is correlated with estimates of overall courtship vigour, particularly in polyandrous males. In total, these results suggest that selection on song pulse rate by females led to the evolution of increased courtship vigour displayed during courtship, indicating a potential correlated response of overall male motor capacities.

Pulse Rate Production

Our results show that pulse rate is not constant over courtship duration, but progressively declines as a male continues to beat his wings. Although this pattern has previously been reported in the courtship songs of two other Drosophila species (D. melanogaster: [START_REF] Bernstein | Temporal analysis of tone pulses within the courtship songs of two sibling Drosophila species, their interspecific hybrid, and behavioral mutants of D. melanogaster (Diptera: Drosophilidae)[END_REF][START_REF] Dow | Function and Organization of Courtship Behaviour in Drosophila melanogaster[END_REF][START_REF] Ewing | Functional aspects of Drosophila courtship[END_REF][START_REF] Wilson | Behavioural pleiotropy of the yellow gene in Drosophila melanogaster[END_REF]D. simulans: Bernstein et al., 1992), its relevance and implications for sexual selection

have not yet been considered. In D. pseudoobscura, pulse rate appears to decrease progressively both within a burst of song, and also along the burst sequence. The pattern observed in our study suggests that males start producing song with a fast pulse rate but cannot sustain this as courtship progresses (and particularly for males who evolved under monogamy conditions). Repetitive signals are thought to provide a useful measure of mate quality to the receivers, both via the average rate at which they are produced and the variation of this rate (i.e. increase or decrease) over courtship time [START_REF] Kotiaho | Energetic costs of size and sexual signalling in a wolf spider[END_REF][START_REF] Mowles | Repetitive signals and mate choice: insights from contest theory[END_REF]. As a song with a fast pulse rate can be a physically challenging task, requiring both sustained motor power and motor endurance and thus pushing males to their maximum capacities, variation in the ability of males to maintain a given pulse rate over time has the potential to accurately reflect mate condition.

Pulse rate production is associated with both temperature and body size variation. The effect of temperature on acoustic signalling is common through its effect on metabolic rate and has already been shown in many species [START_REF] Gillooly | Effects of size and temperature on metabolic rate[END_REF][START_REF] Gillooly | The energetic basis of acoustic communication[END_REF], including Drosophila species [START_REF] Noor | Courtship songs of Drosophila pseudoobscura and D. persimilis: analysis of variation[END_REF][START_REF] Ritchie | Rapid evolution of courtship song pattern in Drosophila willistoni sibling species[END_REF][START_REF] Ritchie | Genetic variability of courtship song in a population of Drosophila melanogaster[END_REF][START_REF] Ritchie | Characterization of female preference functions for Drosophila montana courtship song and a test of the temperature coupling hypothesis[END_REF]. In addition to this effect, we show that temperature is not only associated with mean pulse rate, but also with pulse rate decrease over time, indicating that both power output and endurance are temperature-dependent. These results suggest that pulse rate probably strongly relies on male physiological state [START_REF] Lailvaux | A functional perspective on sexual selection: insights and future prospects[END_REF].

Pulse rate also positively correlates with body size, this effect being unsurprising as motor power often positively covaries with body size [START_REF] Biewener | Animal locomotion[END_REF][START_REF] Carrier | Ontogenetic limits on locomotor performance[END_REF]. The influence of body size on pulse rate has rarely been investigated in Drosophila species, sometimes only via correlations between body size and 'raw' pulse rate data (i.e. not temperature-corrected), which failed to find an association between size and rate [START_REF] Hoikkala | Male courtship song frequency as an indicator of male genetic quality in an insect species, Drosophila montana[END_REF][START_REF] Partridge | Male size and mating success in Drosophila melanogaster : the roles of male and female behaviour[END_REF]. The positive influence of body size on pulse rate found here indicates that larger males are able to produce a faster pulse rate than smaller males, suggesting that body size influences motor power. This effect could potentially be due to variation in thoracic muscle size, and lead to a higher power output (i.e. a faster pulse rate) of larger males. Indeed, thorax volume is positively correlated with flight wing-beat frequency in D. melanogaster [START_REF] Curtsinger | Genetic variability of flight metabolism in Drosophila melanogaster. I. Characterization of power output during tethered flight[END_REF]. Overall, these results suggest pulse rate has the ability to reflect both male motor power and endurance to females, potentially making it an evolutionary driver of overall male motor capacities [START_REF] Clark | The role of power versus energy in courtship: what is the "energetic cost" of a courtship display?[END_REF].

Effect of Sexual Selection on Courtship Song Evolution

Males from polyandrous lines are not only more likely to produce song and produce song faster, but they are also able to maintain a fast rate for longer than males from the monogamous lines, demonstrating that manipulating sexual selection had a significant impact on male motor performance during courtship. Our results are consistent with previous work suggesting that polyandrous females prefer faster male pulse rates [START_REF] Debelle | Evolution of divergent female mating preference in response to experimental sexual selection[END_REF][START_REF] Williams | Courtship songs of Drosophila pseudoobscura and D. persimilis. II. Genetics of species differences[END_REF]. Signals with an increased energy content have been shown to be under directional female preference in several species (e.g. in frogs: [START_REF] Gerhardt | Experimental analysis of multivariate female choice in gray treefrogs (Hyla versicolor): evidence for directional and stabilizing selection[END_REF][START_REF] Ryan | Energy, calling, and selection[END_REF]; in wolf spiders: Shamble, Wilgers, Swoboda, & Hebets, 2009; in crickets: Simmons, Thomas, Simmons, & Zuk, 2013), with females typically preferring louder song, higher calling rate and higher pulse repetition rate [START_REF] Clark | The role of power versus energy in courtship: what is the "energetic cost" of a courtship display?[END_REF][START_REF] Mowles | Repetitive signals and mate choice: insights from contest theory[END_REF]. The fact that the ability to sustain a fast pulse rate has been affected by sexual selection manipulation suggests that selection by females towards fast pulse rates led to the evolution of males delivering songs with increased motor power and sustained intensity (i.e. more endurance), and indicates that pulse rate may be used as an indicator of male motor performance by females.

We find, after c.a. 110 generations of selection, a difference in average pulse rate in the same direction than in the preliminary song study (conducted after 30 generations of selection; [START_REF] Snook | Experimental manipulation of sexual selection and the evolution of courtship song in Drosophila pseudoobscura[END_REF]. The comparable difference in pulse rate between males from polyandrous and monogamous lines after a further 80 generations of selection (1.54 ms between the polyandry and monogamy treatments in [START_REF] Snook | Experimental manipulation of sexual selection and the evolution of courtship song in Drosophila pseudoobscura[END_REF]1.57 ms in the current study, see Table 2) could indicate that pulse rate evolution has reached stable equilibrium conditions between sexual and viability selection [START_REF] Hine | Natural selection stops the evolution of male attractiveness[END_REF][START_REF] Kirkpatrick | Good genes and direct selection in the evolution of mating preferences[END_REF], but could also mean that genetic variation for faster pulse rates has been depleted in the polyandrous lines. Two studies using artificial selection on pulse rate in D. melanogaster showed a lower evolutionary response towards faster pulse rates [START_REF] Ritchie | Artificial selection for a courtship signal in Drosophila melanogaster[END_REF][START_REF] Turner | Investigating natural variation in Drosophila courtship song by the evolve and resequence approach[END_REF], suggesting reduced expressed genetic variation for fast pulse rates in this environment, an expected result if selection has persistently acted in this direction.

Male body size responded to the variation in sexual selection opportunity, with males from the polyandrous lines being overall larger than males from the monogamous lines. Body size commonly responds to pre-copulatory sexual selection among species [START_REF] Andersson | Sexual selection[END_REF][START_REF] Blanckenhorn | The evolution of body size: what keeps organisms small?[END_REF][START_REF] Thornhill | The evolution of insect mating systems[END_REF] and affects male mating success in several Drosophila species (including D. pseudoobscura), with larger males winning more aggressive encounters, delivering more courtship and mating faster [START_REF] Ewing | Body size and courtship behaviour in Drosophila melanogaster[END_REF][START_REF] Partridge | Male size and mating success in Drosophila melanogaster and D. pseudoobscura under field conditions[END_REF]Partridge et al., 1987;[START_REF] Partridge | Lifetime mating success of male fruitflies (Drosophila melanogaster) is related to their size[END_REF]. As body size also influences pulse rate, any pulse rate difference between the experimental evolution treatments could thus be explained by size differences. However, even after controlling for the effects of body size (cf. Appendix A and D), sexual selection treatments still differ in their pulse rate production pattern, indicating that traits other than body size have diverged between the treatments and contribute to the differences in motor signalling between the treatments.

Mate Choice As An Evolutionary Mechanism Driving The Evolution Of Motor Performance

Males from polyandrous lines are more vigorous than males from monogamous lines. Indeed, males from polyandrous lines have an enhanced mating capacity and a higher courtship frequency relative to males from monogamous lines [START_REF] Crudgington | Increased opportunity for sexual conflict promotes harmful males with elevated courtship frequencies[END_REF][START_REF] Crudgington | Experimental manipulation of sexual selection promotes greater male mating capacity but does not alter sperm investment[END_REF]. Our study also shows that males from polyandrous lines start producing song faster, produce a faster pulse rate, and have a higher endurance than males from monogamous lines. In theory, male-male competition could participate to this observed increase in male motor capacities. A direct effect of male-male competition on the evolution of pulse rate seems however unlikely, as courtship song is a near-field sound [START_REF] Bennet-Clark | Acoustics of insect song[END_REF], 1998) and pulse rate value does not affect other males behaviour [START_REF] Talyn | The role of courtship song in sexual selection and species recognition by female Drosophila melanogaster[END_REF]. Conversely, pulse rate affects male mating success in nochoice assays in this species [START_REF] Williams | Courtship songs of Drosophila pseudoobscura and D. persimilis. II. Genetics of species differences[END_REF], pulse rate coevolved with female preference for pulse rate in our experimental lines [START_REF] Debelle | Evolution of divergent female mating preference in response to experimental sexual selection[END_REF], and pulse rate is correlated with other courtship motor traits. This suggests that the action of mate choice on pulse rate is actively involved in the observed evolutionary motor changes in our experimental lines.

Drosophila courtship song has so far only been linked to non-motor selective benefits (i.e. high intrapulse frequency in D. montana is associated with a higher male mating success and a higher offspring survival; [START_REF] Hoikkala | Male courtship song frequency as an indicator of male genetic quality in an insect species, Drosophila montana[END_REF][START_REF] Ritchie | Characterization of female preference functions for Drosophila montana courtship song and a test of the temperature coupling hypothesis[END_REF]. Our results support the idea that Drosophila courtship song could also signal motor performance. Our analysis focused on courtship-related traits, however motor performance expressed during courtship is likely to reflect an individual's overall motor performance [START_REF] Byers | Female mate choice based upon male motor performance[END_REF][START_REF] Clark | The role of power versus energy in courtship: what is the "energetic cost" of a courtship display?[END_REF][START_REF] Lailvaux | A functional perspective on sexual selection: insights and future prospects[END_REF][START_REF] Oufiero | Evaluating performance costs of sexually selected traits[END_REF]. In a context of strong sexual selection, the selection of fast singing males by females could thus also influence the evolution of other motor characteristics (e.g. flying ability, competitive ability, etc.; [START_REF] Byers | Female mate choice based upon male motor performance[END_REF].

Contrary to what we observe in the polyandrous lines, reducing the opportunity for sexual selection in the monogamous lines is associated with lower singing probability, a longer singing latency, and the inability to maintain a fast pulse rate. This suggests that these traits are costly and could be selected against in the absence of mating competition. Males from monogamous lines also have a lower courtship frequency compared to males from polyandrous lines [START_REF] Crudgington | Increased opportunity for sexual conflict promotes harmful males with elevated courtship frequencies[END_REF]. As courting (without mating) has been shown to reduce male longevity in D. melanogaster [START_REF] Cordts | Courtship reduces longevity of male Drosophila melanogaster[END_REF], these results overall suggest that intense courtship song could impose an important fitness cost to males in a monogamous context, which may have resulted in the reduced investment in courtship song observed in populations under relaxed sexual selection [START_REF] Crudgington | Increased opportunity for sexual conflict promotes harmful males with elevated courtship frequencies[END_REF][START_REF] Crudgington | Experimental removal and elevation of sexual selection: does sexual selection generate manipulative males and resistant females[END_REF].

In conclusion, our results suggest that the pulse rate has the potential to be an indicator of male condition to females, and that the action of female choice on this motor signal affected male motor performance during courtship in our replicated experimental populations. In natural populations, female selection of male courtship motor performance could thus have an impact on the evolution of motor performance exhibited in contexts other than courtship. This work contributes to the limited number of studies providing evidence that sexual selection via mate choice of motor signals may also drive the evolution of mate motor performance [START_REF] Byers | Female mate choice based upon male motor performance[END_REF][START_REF] Mowles | Repetitive signals and mate choice: insights from contest theory[END_REF]. Further work in this and other systems should quantify the selective benefits gained by an increased motor performance in mating and non-mating contexts (e.g. standard locomotion, foraging, escaping predators), and investigate what evolutionary changes lead to enhanced motor signals (e.g. morphological, anatomical, physiological), to gain a better understanding of the influence of sexual selection on the evolution of motor performance.

LRR song was analysed similarly to HRR song. To allow distinguishing between interpulse and interburst intervals, an upper threshold was also determined visually by plotting the distribution of the duration between two pulses (LRR threshold = 482ms, the mean LRR interpulse interval is approximately 220 ms in our populations; [START_REF] Snook | Experimental manipulation of sexual selection and the evolution of courtship song in Drosophila pseudoobscura[END_REF]

Differences In Singing Probability Between The Sexual Selection Treatments

The difference in the probability of singing LRR (i.e. the probability of a male producing at least a single pulse of LRR) between the sexual selection treatments was investigated using a Fisher's exact test on the pooled replicates, due to the distribution of LRR data (as polyandrous males always produced LRR in all replicates, but monogamous males do not, model parameters could not be estimated by a linear model as maximum likelihood estimates do not exist for this particular pattern of data; [START_REF] Albert | On the existence of maximum likelihood estimates in logistic regression models[END_REF].

Multivariate Response Of Courtship Song To Sexual Selection Treatment

LRR interpulse interval does not vary over the length of courtship (see Table A1), and therefore values were averaged over the entire length of each recording, and the resulting mean LRR interpulse interval was used for statistical modelling. We fitted a multivariate LMM on LRR song traits with the same structure as the one for HRR song, to test for a response of the mean LRR interpulse interval (of the entire recording in this case), the mean LRR intrapulse frequency, the total number of LRR pulses produced and the LRR singing latency to sexual selection manipulation (Table A2). The number of estimated fixed-effect parameters were 12 ([1+2]x4), with [4+6]x2 = 20 (co)variance parameters. We also estimated the variance between replicates separately for each trait (4 variances) as random effects.

We also tested the significance of correlations between song traits (i.e. LRR interpulse interval, LRR intrapulse frequency, the total number of LRR pulses produced and LRR latency), as well as the differences in song trait correlations between sexual selection treatments.

The same model was also run while including individual body size as a covariate (Table A3).

The number of estimated fixed-effect parameters was this time 16 ([1+1+2]x4), with [4+6]x2 = 20 (co)variance parameters.

Results

Differences In LRR Singing Probability Between The Sexual Selection Treatments

The probability of singing LRR song differs between treatments, with monogamous males having a lower probability than polyandrous males (Fisher's exact test; polyandrous males: 0 recordings without LRR song out of 231 recordings; monogamous males: 11 recordings without LRR song out of 230 recordings; P<0.001).

Evolutionary response to sexual selection manipulation

The multivariate LMM does not identify any significant response of LRR traits to selection sexual treatment (Table A2).

Temperature: The multivariate LMM shows that LRR interpulse interval shortens with temperature (Table A3). Increasing temperature by one degree reduces the interpulse interval by 3.28ms (95% C.I. = -0.67 --6.05, P=0.02). Both LRR singing latency and LRR intrapulse frequency significantly increase with temperature, with an increase in temperature of one degree resulting in a latency increase of 14.5s (95% C.I. = 302 -2558, P=0.01), and an LRR intrapulse frequency increase of 4.18Hz (95% C.I. = 1.20 -7.18, P=0.01).

Body Size: Including body size in the multivariate LMM does not change the results of the model (Table A3). LRR interpulse interval shortens with body size, with an increase of 1 standard deviation in wing size being associated with an interpulse interval reduction of 4.72ms (95% C.I. = -1.52--7.53, P=0.004).

Analysis of the Associations Between Courtship traits

The multivariate LMM also revealed that LRR interpulse interval was positively correlated with the total number of LRR pulses produced (Table A4), but the correlation reached significance only for monogamous males. As with HRR song, we found a significant negative association between LRR latency and the total number of LRR pulses produced for both treatments (Table A4). There was no significant impact of the sexual selection treatment on the correlations between LRR traits (Table A5, Fig. A1).

Including body size in the multivariate LMM generated very similar results (Tables A6,A7). In both models, sexual selection treatment was tested as a fixed effect, and replicate was included as a random effect. The following elements are specified: the model estimate of each variable (β), the lower and upper limit of the estimate's 95% confidence interval (CI), and p-value of the test comparing the estimate to zero (P). HRR = high-repetition rate song, Treatment = sexual selection treatment (E = polyandrous; the monogamous treatment M was used as the reference level). N = 471 recordings for HRR probability and N = 355 recordings for body size. * Given that the HRR singing probability GLMM used a binomial error distribution, the given estimates for this model are on a logit scale. The following elements are specified: the model estimate of each variable (β), the lower D1). The following elements are specified: the correlation coefficient (r) and the p-value (P). LRR = low-repetition rate song, IPI = interpulse interval, Latency = the time taken to sing the first pulse of LRR song. These correlations were derived from the variances and covariances estimated by the multivariate LMM (see Table A2). Since correlation matrices are symmetric, correlation values for polyandrous males and monogamous males are shown above and below the diagonal, respectively. The following elements are specified: the corresponding correlation coefficients for polyandrous (r E ) and monogamous males (r M ) and the p-value (P). LRR = low-repetition rate song, IPI = interpulse interval, Latency = the time taken to sing the first pulse of LRR song. The following elements are specified: the correlation coefficient (r) and the p-value (P). LRR = low-repetition rate song, IPI = interpulse interval, Latency = the time taken to sing the first pulse of LRR song. These correlations were derived from the variances and covariances estimated by the multivariate LMM (see Table A3). Since correlation matrices are symmetric, correlation values for polyandrous males and monogamous males are shown above and below the diagonal, respectively. The following elements are specified: the model estimate of each variable (β), the lower and upper limit of the estimate's 95% confidence interval (CI), the p-value of the test comparing The following elements are specified: the corresponding correlation coefficients for polyandrous (r E ) and monogamous males (r M ) and the p-value (P). HRR = high-repetition rate song, IPI = interpulse interval, Bursts = the total number of HRR bursts produced, Latency = the time taken to sing the first burst of HRR song. and HRR (high-repetition rate) song are represented. Each burst of song is composed of multiple pulses, each separated by a certain time interval, the interpulse interval (IPI; i.e. the inverse of pulse rate). As the interpulse interval represents the amount of time between two consecutive pulses, a short interpulse interval means that a male rapidly beats his wings (i.e.

FIGURES

fast pulse repetition rate), whereas that a long interpulse interval means that a male slowly beats his wings (i.e. slow pulse repetition rate). HRR frequency represents the intrapulse frequency of a pulse of HRR. In our analysis, we will refer to 'burst position' as the position of an HRR burst in the courtship sequence (i.e. the 1 st burst produced, the 2 nd burst produced, the 3 rd burst produced), and to 'pulse position' as the position of a pulse within a burst of HRR (i.e. the 1 st pulse of a burst, the 2 nd pulse of a burst, the 3 rd pulse of a burst). A4. The dotted light grey circle represents a null correlation (r = 0).

The stronger the correlation, the narrower the ellipse becomes.

  and upper limit of the estimate's 95% confidence interval (CI), and the p-value of the test comparing the estimate to zero (P). BP = Burst position, PP = Pulse position, HRR = highrepetition rate song, IPI = interpulse interval, Treatment = sexual selection treatment (E = polyandrous; the monogamous treatment M was used as the reference level), burst position = the position of the burst in the recording, pulse position = the position of the pulse in the HRR burst. The autocorrelation parameters are φ 1 = 0.23, φ 2 = 0.12, φ 3 = 0.058 and φ 4 = 0.031. N = 35206 individual interpulse interval values. The same model was fitted while including body size as a covariate (Table

  the estimate to zero (P). BP = Burst position, PP = Pulse position, HRR = high-repetition rate song, IPI = interpulse interval, Treatment = sexual selection treatment (E = polyandrous; the monogamous treatment M was used as the reference level), burst position = the position of the burst in the recording, pulse position = the position of the pulse in the HRR burst. The autocorrelation parameters are φ 1 = 0.23, φ 2 = 0.12, φ 3 = 0.058 and φ 4 = 0.031. N = 35206 individual interpulse interval values.

Figure 1 .

 1 Figure 1. D. pseudoobscura courtship song representation. Both LRR (low-repetition rate)

Figure 2 .Figure 3 .Figure 4 .Figure 5 .Figure 6 .Figure A1 .

 23456A1 Figure 2. Differences between the sexual selection treatments in singing probability (the

Figure B1 .

 B1 Figure B1. Example of HRR interpulse interval lengthening along HRR bursts, over courtship

Figure B2 .

 B2 Figure B2. Violin plots showing the distribution of: a) burst number and b) pulse number

Table 1 .

 1 Summary tables for the fitted GLMM analysing HRR singing probability and the univariate LMM analysing body size.

			HRR singing probability	Body size
	Model	Factor	Lower	Upper	Lower	Upper
			β *	P	β	P
	parameters	level	CI	CI	CI	CI
	Treatment	E	1.20 0.56	1.85 <0.001 0.023 0.010 0.035 <0.001
	Intercept		1.35 0.98	1.71 <0.001	1.08 1.07	1.09 <0.001
	Inter-replicate variance	0.038	0.000066
	Residual variance		-	0.00062

Table 2 .

 2 Summary table for the fitted univariate LMM analysing the HRR pulse rate production between and within HRR bursts.

		Factor		Lower
	Model parameters		β	Upper CI	P
		level		CI
	Treatment	E	-1.57	-2.64	-0.501	0.026
	Temperature		-0.71	-0.81	-0.62	<0.001
	BP		0.69	0.51	0.87	<0.001
	PP		0.92	0.87	0.98	<0.001
	Treatment * BP		-0.10	-0.32	0.11	0.339
	Temperature * BP		-0.30	-0.40	-0.20	<0.001
	Treatment * PP		-0.093	-0.17	-0.018	0.013
	BP * PP		0.11	0.039	0.17	0.002
	Treatment * BP * PP		-0.16	-0.24	-0.076	<0.001
	Intercept		39.27	38.52	40.01	<0.001
	Inter-replicate variance			0.56
	Inter-burst variance (nested within
				3.47
	replicate)
	Residual variance			4.92

Table 3 .

 3 Summary table for the fitted multivariate LMM analysing HRR traits. Latency = the time taken to sing the first pulse of LRR song. Covariances between the response variables of the model are provided as correlations in TableA6.Estimated variances between replicates were σ 2 = 0.23 for IPI, σ 2 = 0.13 for intrapulse frequency, σ 2 = 0.01 for the total number of bursts and σ 2 = 0.06 for latency. Note that all responses are expressed as z-scores of the log transformed value of the original measurements. Wing size was also transformed into z-scores, but temperature was not altered. Estimates in the table are thus not expressed on the original data scale, but in zscores of log values (see Methods). The means and standard deviations of the log of the original variables are as following: IPI (mean = 5.34, σ = 0.14), frequency (mean = 6.19, σ =

			Factor
	Trait	Model parameters		β	Lower CI Upper CI	P
			level
		Treatment	E	-0.49	-1.10	0.12	0.092
	IPI	Temperature		-0.14	-0.24	-0.028	0.012
		Intercept		3.41	1.14	6.10	0.010
		Treatment	E	-0.21	-0.57	0.18	0.262
	Amplitude	Temperature		0.083	-0.032	0.188	0.158
		Intercept		-1.79	-4.24	0.78	0.176
		Treatment	E	-0.196	-0.771	0.43	0.446
	Frequency	Temperature		0.076	-0.024	0.20	0.172
		Intercept		-1.65	-4.20	0.89	0.218
		Treatment	E	0.21	-0.31	0.74	0.398
	Total number of
		Temperature		0.047	-0.078	0.15	0.414
	bursts
		Intercept		-1.16	-3.84	1.45	0.378
		Treatment	E	-0.51	-0.77	-0.25	0.001
	Latency	Temperature		0.080	-0.061	0.19	0.192
		Intercept		-1.56	-4.25	1.39	0.254
	The following elements are specified: the model estimate of each variable (β; here the
	posterior mean), the lower and upper limits of the estimate's 95% credibility interval (CI),
	and the p-value of the test comparing the estimate to zero (P). IPI = interpulse interval,

Table A4 .

 A4 Correlation matrix between LRR song traits for the two sexual selection treatments.

		IPI		Frequency	Pulse number	Latency
		r	P	r	P	r	P	r	P
	IPI	1	-	-0.14	0.088	0.07	0.38	0.06	0.428
	Frequency	-0.14	0.06	1	-	0.06	0.466	-0.01	0.900
	Pulse
		0.22	0.002	0.03	0.650	1	-	-0.23	0.001
	number
	Latency	0.07	0.358	-0.06	0.512	-0.35	<0.001	1	-

Table A5 .

 A5 Differences in LRR song trait correlations between the sexual selection treatments (r E -r M from TableA4).

		IPI		Frequency	Pulse number	Latency
		r E -r M	P	r E -r M	P	r E -r M	P	r E -r M	P
	IPI	-	-
	Frequency	0.00	0.988	-	-
	Pulse number	-0.15	0.152	0.03	0.802	-	-
	Latency	-0.01	0.978	0.04	0.684	0.12	0.224	-	-

Table A6 .

 A6 Correlation matrix between LRR song traits for the two sexual selection treatments

	(with body size included in the model)
		IPI		Frequency	Pulse number	Latency
		r	P	r	P	r	P	r	P
	IPI	1	-	-0.13	0.100	0.09	0.238	0.06	0.450
	Frequency	-0.15	0.052	1	-	0.06	0.482	-0.01	0.900
	Pulse
		0.21	0.004	0.03	0.682	1	-	-0.23	0.004
	number
	Latency	0.09	0.256	-0.05	0.556	-0.35	<0.001	1	-

Table A7 .

 A7 Differences in LRR song trait correlations between the sexual selection treatments (r E -r M from TableA6) (with body size included in the model)

		Inter-replicate variance						0.73
	1008	production between and within HRR bursts (with body size included in the model).
		Inter-burst variance (nested within replicate)				3.32
			IPI		Frequency Factor	Pulse number	Latency
		Model parameters			β	Lower CI Upper CI	P
		Residual variance				level			4.91
			r E -r M	P	r E -r M	P	r E -r M	P	r E -r M	P
		Wing size					-0.42	-0.53	-0.31 <0.001
		IPI	-	-
		Treatment				E	-1.17	-2.39	0.046	0.102
		Frequency	0.01	0.868	-	-
		Temperature					-0.75	-0.85	-0.65 <0.001
		Pulse number	-0.12	0.262	0.02	0.886	-	-
		BP					0.69	0.51	0.87	<0.001
		Latency	-0.03	0.808	0.04	0.722	0.12	0.252	-	-
		PP					0.92	0.86	0.98	<0.001
		The following elements are specified: the corresponding correlation coefficients for
		polyandrous (r E ) and monogamous males (r M ) and the p-value (P). LRR = low-repetition rate Treatment * BP -0.055 -0.27 0.16 0.609
		song, IPI = interpulse interval, Latency = the time taken to sing the first pulse of LRR song.
		Temperature * BP					-0.3	-0.40	-0.20 <0.001
		Treatment * PP					-0.09	-0.16	-0.015	0.015
		BP * PP					0.11	0.042	0.18	0.001
		Treatment * BP * PP				-0.16	-0.24	-0.079 <0.001
		Intercept					39.04	38.20	39.89 <0.001

Table D2 .

 D2 Summary table for the fitted multivariate LMM analysing HRR song (with body size 1010

	1011	included in the model)
		Song trait	Model parameters Factor level	β	Lower CI Upper CI	P
			Treatment	E	-0.41	-1.014	0.26	0.178
			Temperature		-0.14	-0.26	-0.045	0.016
		IPI
			Body size		-0.13	-0.27	-0.014	0.046
			Intercept		3.51	1.032	5.89	0.008
			Treatment	E	-0.32	-0.74	0.11	0.128
			Temperature		0.095	-0.018	0.21	0.106
		Amplitude
			Body size		0.16	0.042	0.30	0.024
			Intercept		-2.02	-4.63	0.57	0.138
		Frequency	Treatment	E	-0.11	-0.63	0.52	0.678

Table D4 .

 D4 Differences in HRR courtship trait correlations between the sexual selection treatments (r E -r M from TableD3) (with body size included in the model).

		IPI		Amplitude	Frequency	Bursts		Latency
		r E -r M	P	r E -r M	P	r E -r M	P	r E -r M	P	r E -r M	P
	IPI	-	-
	Amplitude	-0.09 0.426	-	-
	Frequency	-0.18 0.142	0.14 0.218	-	-
	Bursts	-0.44 <0.001 0.14 0.290 0.25 0.042	-	-
	Latency	0.31 0.012 -0.09 0.484 -0.05 0.700 0.10 0.360	-	-

Figure B1 shows a pattern in pulse rate production over courtship time, by illustrating how HRR interpulse interval lengthens as courtship time increases. Figure B2 shows the range of the distribution of the burst and pulse numbers, depending on sexual selection treatment and recording temperature.

APPENDIX C -Prior Definition For Fitting The Multivariate LMM.

In both MCMCglmm models (the one with body size and the one without), we retained the default settings for the prior distributions for fixed effects. In contrast, we set identity matrices as prior specification for all other prior distributions and we used a degree of belief of 0.001 for the priors used in variance estimations only (i.e. weakly informative improper prior) and a degree of belief equal to the number of response variables plus one (i.e. proper prior) for the prior used in covariance matrix estimations. This structure follows the recommendations of the package instructions.

APPENDIX D -HRR Song Analyses (With Body Size Included)

Running analyses of HRR interpulse interval while including body size as a covariate shows that, although body size has a significant effect on courtship song production, the effect of sexual selection treatment also remains significant (see Tables D1-4).

Treatment = sexual selection treatment (E = polyandrous; the monogamous treatment M was used as the reference level), Latency = the time taken to sing the first burst of HRR song. Covariances between the response variables of the model are provided as correlations in Table 4. Estimated variances between replicates were σ 2 = 0.17 for IPI, σ 2 = 0.04 for amplitude, σ 2 = 0.14 for intrapulse frequency, σ 2 = 0.12 for the total number of bursts and σ 2 = 0.01 for latency. Note that all responses are expressed as z-scores of the log transformed value of the original measurements, but temperature was not altered.

Estimates in the table are thus not expressed on the original data scale, but in z-scores of log values (see Methods). The means and standard deviations of the log of the original variables are as following: IPI (mean = 3.63, σ = 0.06), amplitude (mean = 5.85, σ = 0.20), frequency (mean = 5.56, σ = 0.09), total number of bursts (mean = 2.35, σ = 0.84), latency (mean = 9.38, σ = 1.32). N = 280 recordings. The same model was fitted while including body size as a covariate (Table D2). The following elements are specified: the correlation coefficient (r) and the p-value (P).

HRR = high-repetition rate song, IPI = interpulse interval, Bursts = the total number of HRR bursts produced, Latency = the time taken to sing the first burst of HRR song. These correlations were derived from the variances and covariances estimated by the multivariate LMM (see Table 3). Since correlation matrices are symmetric, correlation values for polyandrous males and monogamous males are shown above and below the diagonal, respectively. A4. Estimated variances between replicates were σ 2 = 0.20 for IPI, σ 2 = 0.12 for intrapulse frequency, σ 2 = 0.01 for the total number of bursts and σ 2 = 0.06 for latency. Note that all responses are expressed as z-scores of the log transformed value of the original measurements, but temperature was not altered. Estimates in the table are thus not expressed on the original data scale, but in z-scores of log values (see Methods).

The means and standard deviations of the log of the original variables are as following: IPI (mean = 5.34, σ = 0.14), frequency (mean = 6.19, σ = 0.06), total number of pulses (mean = 3.36, σ = 0.78), latency (mean = 8.90, σ = 1.38). N = 415 recordings. The same model was fitted while including body size as a covariate (Table A3). The following elements are specified: the model estimate of each variable (β; here the 1012 posterior mean), the lower and upper limits of the estimate's 95% credibility interval (CI), and 1013 the p-value of the test comparing the estimate to zero (P). IPI = interpulse interval, Treatment = sexual selection treatment (E = polyandrous; the monogamous treatment M was used as the reference level), Latency = the time taken to sing the first burst of HRR song. Covariances between the response variables of the model are provided as correlations in Table D3.

Estimated variances between replicates were σ 2 = 0.2 for IPI, σ 2 = 0.05 for amplitude, σ 2 = 0.13 for intrapulse frequency, σ 2 = 0.12 for the total number of bursts and σ 2 = 0.01 for latency. Note that all responses are expressed as z-scores of the log transformed value of the original measurements. Wing size was also transformed into z-scores, but temperature was not altered. Estimates in the table are thus not expressed on the original data scale, but in z- The following elements are specified: the correlation coefficient (r) and the p-value (P). HRR = high-repetition rate song, IPI = interpulse interval, Bursts = the total number of HRR bursts produced, Latency = the time taken to sing the first burst of HRR song. These correlations were derived from the variances and covariances estimated by the multivariate LMM (see Table D2). Since correlation matrices are symmetric, correlation values for polyandrous males and monogamous males are shown above and below the diagonal, respectively.