Sandra Goutte
email: s.m.goutte@gmail.com

Alain Dubois

Samuel D Howard

Rafael Marquez

Jodi J L Rowley

J Maximilian Dehling

Philippe Grandcolas

Xiong Rongchuan

Frédéric Legendre

Fr Éd Éric Legendre

Environmental constraints and call evolution in torrent-dwelling frogs

Keywords: Acoustic adaptation hypothesis, ancestral state reconstruction, Anura, noisy environments, phylogeny, Ranidae

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The evolution of amphibian vocal organs has been mentioned as early as in Darwin's "Descent of Man" [START_REF] Darwin | The descent of man, and selection in relation to sex[END_REF]) but the forces driving the evolution of vocalizations remain poorly understood. A multitude of factors could be shaping these acoustic signals, complicating the investigation of their evolution. Natural (environmental and predator-driven) and sexual (inter-and intra-) selection and phylogenetic inertia may act in opposite and sometimes surprising directions. In addition, body size (e.g., [START_REF] Zimmerman | A comparison of structural features of calls of open and forest Habitat frog species in the central Amazon[END_REF][START_REF] Ryan | Selection on long-distance acoustic signals[END_REF][START_REF] Hoskin | Ecology and taxonomydriven deviations in the frog call-body size relationship across the diverse Australian frog fauna[END_REF][START_REF] Gingras | Call acoustics reflect body size across four clades of anurans[END_REF]) and physiological factors, such as body temperature (Gerhardt 1978;[START_REF] Gayou | Effects of temperature on the mating call of Hyla versicolor[END_REF][START_REF] Marquez | Advertisement calls of the midwife toads Alytes (Amphibia, Anura, Discoglossidae) in continental Spain[END_REF][START_REF] Navas | Thermal dependency of calling performance in the eurythermic frog Colostethus subpunctatus[END_REF][START_REF] Bevier | Variation in call structure and calling behavior in treefrogs of the genus Scinax[END_REF] or hormonal level [START_REF] Penna | Hormone-induced vocal behavior and midbrain auditory sensitivity in the green treefrog, Hyla cinerea[END_REF][START_REF] Boyd | Arginine vasotocin facilitation of advertisement calling and call phonotaxis in bullfrogs[END_REF][START_REF] Leary | Steroid hormone profiles and relative body condition of calling and satellite toads: implications for proximate regulation of behavior in anurans[END_REF]), affect some characteristics of the vocalizations and might induce analytical biases if they are not considered [START_REF] Zimmerman | A comparison of structural features of calls of open and forest Habitat frog species in the central Amazon[END_REF][START_REF] Bevier | Variation in call structure and calling behavior in treefrogs of the genus Scinax[END_REF][START_REF] Erdtmann | Environmental effects on anuran call design: what we know and what we need to know[END_REF]. These challenges have resulted in relatively few papers on frog vocalizations within a phylogenetic context [START_REF] Ryan | Sexual selection and signal evolution: the ghost of biases past[END_REF][START_REF] Cocroft | Patterns of advertisement call evolution in toads and chorus frogs[END_REF][START_REF] Cannatella | Phylogeny of frogs of the Physalaemus pustulosus species group, with an examination of data incongruence[END_REF][START_REF] Lodé | Character convergence in advertisement call and mate choice in two genetically distinct water frog hybridogenetic lineages (Rana kl esculenta, Rana kl grafi)[END_REF][START_REF] Robillard | Evolution of advertisement signals in North American hylid frogs: vocalizations as endproducts of calling behavior[END_REF][START_REF] Wollenberg | Molecular phylogeny of Malagasy reed frogs, Heterixalus, and the relative performance of bioacoustics and color-patterns for resolving their systematics[END_REF][START_REF] Goicoechea | Recovering phylogenetic signal from frog mating calls[END_REF][START_REF] Gingras | Call acoustics reflect body size across four clades of anurans[END_REF], where studies are often limited to few species and small phylogenetic groups.

Environmental pressure acts as one of the major selective forces on acoustic signals' evolution [START_REF] Morton | Ecological sources of selection on avian sounds[END_REF][START_REF] Marten | Sound transmission and its significance for animal vocalization[END_REF][START_REF] Wiley | Physical constraints on acoustic communication in the atmosphere: implications for the evolution of animal vocalizations[END_REF][START_REF] Endler | Signals, signal conditions, and the direction of evolution[END_REF][START_REF] Schluter | The ecology of adaptive radiation[END_REF][START_REF] Ey | The "acoustic adaptation hypothesis" -a review of the evidence from birds, anurans and mammals[END_REF][START_REF] Weir | The role of ecological constraint in driving the evolution of avian song frequency across a latitudinal gradient[END_REF]. The physical features of the environment affect both sound transmission and its content integrity, favoring different calls in different environments. As a call that does not reach its intended receiver-or is too distorted to be efficiently recognizable or localizable-often reduces the fitness of the sender, the acoustic properties of a calling site are expected to constrain call evolution and might lead to convergent calls in similar environments. This idea is referred to as the Acoustic Adaptation Hypothesis or AAH [START_REF] Morton | Ecological sources of selection on avian sounds[END_REF][START_REF] Marten | Sound transmission and its significance for animal vocalization[END_REF][START_REF] Wiley | Physical constraints on acoustic communication in the atmosphere: implications for the evolution of animal vocalizations[END_REF][START_REF] Endler | Signals, signal conditions, and the direction of evolution[END_REF][START_REF] Ey | The "acoustic adaptation hypothesis" -a review of the evidence from birds, anurans and mammals[END_REF][START_REF] Weir | The role of ecological constraint in driving the evolution of avian song frequency across a latitudinal gradient[END_REF]).

In the AAH framework, several environmental variables have been highlighted as relevant, for example, the vegetation density of a site or the ambient noise [START_REF] Castellano | Call degradation in diploid and tetraploid green toads[END_REF][START_REF] Ziegler | Linking amphibian call structure to the environment: the interplay between phenotypic flexibility and individual attributes[END_REF]. A high level of ambient noise can be considered a strong selective pressure, as ambient noise can mask the signal if their respective frequency ranges overlap [START_REF] Brumm | Acoustic communication in noise[END_REF]. In such situations, a frequency shift of the signal reducing the masking effect would be advantageous for the sender [START_REF] Brumm | Acoustic communication in noise[END_REF][START_REF] Nemeth | Blackbirds sing higher-pitched songs in cities: adaptation to habitat acoustics or side-effect of urbanization?[END_REF][START_REF] Cunnington | Plasticity in the vocalizations of anurans in response to traffic noise[END_REF][START_REF] Boonman | Evolution of high-frequency communication in frogs[END_REF][START_REF] Cardoso | On the relation between loudness and the increased song frequency of urban birds[END_REF][START_REF] Both | Biological invasions and the acoustic niche: the effect of bullfrog calls on the acoustic signals of white-banded tree frogs[END_REF][START_REF] Goodwin | Shift of song frequencies in response to masking tones[END_REF]. Other acoustic parameters of vocalizations have also been suggested as sensitive to environmental pressures such as rhythm, frequency, and amplitude modulation (AM; Dubois 1977aDubois ,1977b;;[START_REF] Dubois | A case of possible vocal convergence between frogs and a bird in Himalayan torrents[END_REF][START_REF] Grosjean | Description of advertisement calls of six species of the genus Chaparana (Ranidae) from Nepal and India[END_REF][START_REF] Penna | Vocal strategies in confronting interfering sounds by a frog from the southern temperate forest, Batrachyla antartandica[END_REF].

Despite several studies in birds, mammals, and anurans, no general adaptive pattern has been shown between vocalizations and environmental properties [START_REF] Morton | Ecological sources of selection on avian sounds[END_REF][START_REF] Dubois | A case of possible vocal convergence between frogs and a bird in Himalayan torrents[END_REF][START_REF] Nemeth | Blackbirds sing higher-pitched songs in cities: adaptation to habitat acoustics or side-effect of urbanization?[END_REF][START_REF] Peters | Long-distance call evolution in the Felidae: effects of body weight, habitat, and phylogeny[END_REF]. This could be due to a shortcoming in environment characterization (Bosh and De La Riva, 2004;[START_REF] Wells | The ecology and behavior of amphibians[END_REF]Ey and Fisher 2009). Calling sites are often simplified into broad habitat categories such as "forest," "stream," or "open habitat." This simplification does not account for the variability within those categories [START_REF] Morton | Ecological sources of selection on avian sounds[END_REF][START_REF] Zimmerman | A comparison of structural features of calls of open and forest Habitat frog species in the central Amazon[END_REF][START_REF] Kime | The transmission of advertisement calls in Central American frogs[END_REF][START_REF] Bosch | Are frog calls modulated by the environment? An analysis with anuran species from Bolivia[END_REF][START_REF] Vargas-Salinas | Abiotic noise, call frequency and stream-breeding anuran assemblages[END_REF], which might lead to misinterpretations or biased results as shown in other broad typologies [START_REF] Grandcolas | Mapping extrinsic traits such as extinction risks or modelled bioclimatic niches on phylogenies: does it make sense at all?[END_REF][START_REF] Doody | Breaking the socialnon-social dichotomy: a role for reptiles in vertebrate social behavior research?[END_REF][START_REF] Legendre | The evolution of social behaviour in Blaberid cockroaches with diverse habitats and social systems: phylogenetic analysis of behavioural sequences[END_REF]. A few studies have attempted to address this issue but never at a large comparative scale (e.g., [START_REF] Castellano | Call degradation in diploid and tetraploid green toads[END_REF][START_REF] Ziegler | Linking amphibian call structure to the environment: the interplay between phenotypic flexibility and individual attributes[END_REF], taxonomic sample size being a major limitation.

Large and reliable phylogenetic hypotheses are not always available. Therefore, most studies testing environmental adaptation on acoustic signals either ignore phylogenetic relationships (e.g., [START_REF] Penna | Frog call intensities and sound propagation in the South American temperate forest region[END_REF][START_REF] Kime | The transmission of advertisement calls in Central American frogs[END_REF] or integrate them by grouping species at different taxonomic levels (e.g., [START_REF] Zimmerman | A comparison of structural features of calls of open and forest Habitat frog species in the central Amazon[END_REF][START_REF] Bosch | Are frog calls modulated by the environment? An analysis with anuran species from Bolivia[END_REF][START_REF] Bevier | Variation in call structure and calling behavior in treefrogs of the genus Scinax[END_REF]. Nevertheless, methods to account for phylogenetic nonindependence of species exist (Felsenstein 1985a;[START_REF] Grafen | The phylogenetic regression[END_REF]) and have been used in a few studies on acoustic adaptations (e.g., [START_REF] Peters | Long-distance call evolution in the Felidae: effects of body weight, habitat, and phylogeny[END_REF]. This approach allows testing for adaptations but ignores the temporal aspect of those adaptations as evolutionary novelties. To account for both phylogenetic relationships among species and the temporal pattern of lineage divergence, ancestral state reconstructions (ASRs) are needed [START_REF] Grandcolas | Testing adaptation with phylogeny: how to account for phylogenetic pattern and selective value together[END_REF][START_REF] Glor | Phylogenetic insights on adaptive radiation[END_REF]. ASR allows testing for the cooccurrence between an evolutionary novelty and a change in the environmental context, which complements comparative approaches considering phylogenetic relationships among species (Hansen 2014).

Herein we design a phylogenetic study, combined with two procedures to describe calling sites ("broad-type" vs. "fine-scale" codings), and assess how characterizing calling sites at a finer scale affects conclusions on both environmental constraints and call evolution. We examine the evolutionary pattern of several parameters of the advertisement call and, though we focus on torrent frogs (sensu [START_REF] Goutte | The importance of ambient sound level to characterise anuran habitat[END_REF], we undertake this study within a larger phylogenetic context. First, we build a molecular phylogeny including species for which we have acoustic and ecological data. We assess the relationship between body size and call characteristics, which could potentially alter our interpretation of the subsequent analyses. Then we estimate the ancestral values of calling site characteristics, using both broad-type and fine-scale coding methods, to delineate the environmental constraints. We test several models of evolution and evolutionary scenarios of calls based on these ASRs. Finally, we estimate the ancestral values of vocalization traits using the best-fitted model of evolution for these traits. We thus aimed at (1) evaluating the impact of a fine-scale coding of habitats to characterize environmental constraints and (2) investigating the role of ecological constraints on call evolution in ranid frogs.

Materials and Methods

TAXON SAMPLE AND FIELD SITES

The family Ranidae (sensu [START_REF] Frost | The amphibian tree of life[END_REF]) is a large group of frogs (377 species, Frost 2015) showing high diversity in ecology and vocalizations [START_REF] Wells | The ecology and behavior of amphibians[END_REF]. Species of the family Ranidae call in ponds, lakes, waterfalls, rivulets, or large rivers. Species from four Ranidae clades call in or in the close vicinity of fastflowing streams: the genera Staurois, Amolops, and Odorrana, and the group formed by three genera Huia, Clinotarsus, and Meristogenys. These six genera do not belong in a single monophyletic group and some are phylogenetically distant [START_REF] Pyron | A large-scale phylogeny of Amphibia including over 2800 species, and a revised classification of extant frogs, salamanders, and caecilians[END_REF], suggesting multiple, independent acquisitions of the torrent-dwelling lifestyle. The family Ranidae is thus an ideal group to test evolutionary questions on communication with regard to environmental constraints.

We focused on torrent frogs but also sampled species associated with other habitats. Data were collected at 11 localities within five regions of Southeast Asia: Preah Vihear province, Cambodia, December 11 to 20, 2010; West Kalimantan province, Indonesia, from June 9 to August 3, 2011; Sichuan, Hunan, and Hainan provinces, China, from June 7 to July 19, 2012; and Sabah province, Malaysia, August 8 to 24, 2012. Detailed locality data and permit numbers can be found in [START_REF] Goutte | The importance of ambient sound level to characterise anuran habitat[END_REF]. No species currently considered as threatened were sampled (IUCN 2015).

We collected a total of 112 individuals of 37 species for which we recorded vocalizations and fine-scale measurements characterizing their calling sites. After recording, we captured specimens by hand and they were euthanized using a chlorobutanol solution. We collected and recorded 18 individuals of nine additional species, but without any precise data about their calling sites. We also collected individuals from 11 species, which were not calling during the period of our fieldwork, in order to complete our molecular dataset. As outgroups, we recorded and collected individuals from 23 species of the families Rhacophoridae, Dicroglossidae, and Microhylidae. This nonexhaustive sampling is unlikely to represent the diversity of these three families, and we will not discuss results for these groups [START_REF] Grandcolas | Mapping characters on a tree with or without the outgroups[END_REF]). We took tissue samples from liver or thigh muscle and stored them in 100% ethanol for subsequent sequencing. We fixed specimens with a 10% formalin solution for 12-24 h before storing in 70% ethanol solution for museum collection purposes. We measured them after fixation to the nearest 0.1 mm with a Swiss Precision Instrument caliper (model: 31-415-3). Specimens are deposited at the Museum Zoologicum Bogoriense (MZB), Indonesia; the Chengdu Institute of Biology (CIB), China; and the Sabah Museum (SPM), Malaysia.

DNA EXTRACTION, AMPLIFICATION, AND

SEQUENCING

DNA from the samples outlined above and additional samples (Table S1) was extracted using Qiagen DNeasy Tissue Kits. Five molecular markers for a total of approximately 3600 bp were selected according to previous works on ranids (e.g., [START_REF] Wiens | Evolutionary and biogeographic origins of high tropical diversity in old world frogs (Ranidae)[END_REF]) and sequence availability on GenBank. We used two mitochondrial markers 12S (810 bp) and 16S (580 bp) and three nuclear markers: tyrosinase (601 bp), rhodopsin (310 bp), and RAG-1 (1256 bp; Table S1). Amplified DNA fragments (methods detailed in Supporting Information) were sent to the Centre National de Séquenc ¸age (Génoscope, Evry, France) for sequencing.

ALIGNMENT AND PHYLOGENETIC ANALYSES

We obtained a total of 300 sequences and additional sequences were downloaded from GenBank (Table S2). We sampled 80.2% of the taxa for the 12S, 93.2% for the 16S, 74.7% for the tyrosinase, 77.8% for the rhodopsin, and 60.5% for RAG-1. Molecular sequences were aligned using MUSCLE version 3.8.31 [START_REF] Edgar | MUSCLE: multiple sequence alignment with high accuracy and high throughput[END_REF]). We selected partitioning scheme and substitution models using PartitionFinder [START_REF] Lanfear | PartitionFinder: Combined selection of partitioning schemes and substitution models for phylogenetic analyses[END_REF]. For each marker, the best model was a General Time Reversible model with a proportion of invariant sites (I) and a gamma distributed rate variation among sites ( ). However, because I and are strongly correlated [START_REF] Sullivan | The effect of taxon sampling on estimating rate heterogeneity parameters of maximumlikelihood models[END_REF]) and using models mixing these parameters could bias their estimation, we used a GTR + model instead. The best partitioning strategy involved two partitions (mitochondrial and nuclear genes separated).

Phylogenetic reconstructions were performed in maximum likelihood (ML) with RAxML version 7.0.4 [START_REF] Stamatakis | RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models[END_REF][START_REF] Stamatakis | A rapid bootstrap algorithm for the RAxML web servers[END_REF]) and under Bayesian inference (BI) with MrBayes version 3.2.0 [START_REF] Ronquist | MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space[END_REF]. We ran separate and combined analyses. In ML, we selected the best tree from 1000 runs computed from 1000 distinct randomized maximum parsimony starting trees. We calculated support values from 1000 bootstrap replicates using the standard bootstrapping algorithm. In BI, we launched two runs with four chains each for 100 million generations, with trees sampled every 10,000 generations. We assessed the burn-in with Tracer version 1.5 [START_REF] Rambaut | Tracer v1.5[END_REF]. The Potential Scale Reduction Factor (PSRF) values were between 0.999 and 1.014, except for the clock rate parameter (1.144), and effective sample sizes (ESS) were above 200 for all parameters, except the clock rate parameter, indicating a good sample from the posterior probability distribution.

We simultaneously estimated divergence times using an invariant gamma rate relaxed-clock analysis, following the method highlighted in [START_REF] Ronquist | MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space[END_REF]. We used two calibration points: the root of our tree and the most recent common ancestor (MRCA) of our ingroup (i.e., Ranidae). Both values are secondary calibrations taken from dating estimates made in [START_REF] Bossuyt | Phylogeny and biogeography of a cosmopolitan frog radiation: late Cretaceous diversification resulted in continent-scale endemism in the family Ranidae[END_REF]. Their accepted age intervals were 98.6-180.8 and 36.1-85.9 Mya for the root and the Ranidae MRCA, respectively. We used a uniform distribution for both calibration points because we did not have enough information to parameterize meaningfully nonuniform calibrations [START_REF] Sauquet | Testing the impact of calibration on molecular divergence times using a fossil-rich group: the case of Nothofagus (Fagales)[END_REF]. We chose these two calibration points because they are outside of our focus group, for which we did not want to constrain dating. The biogeographic events and fossils used in [START_REF] Bossuyt | Phylogeny and biogeography of a cosmopolitan frog radiation: late Cretaceous diversification resulted in continent-scale endemism in the family Ranidae[END_REF] were inappropriate to our taxon sample and no other data were available, so we relied on secondary calibrations. To calculate the clock rate and branch length rate variance priors used in the relaxed-clock analysis, we first ran a nonclock analysis, thereby getting a starting topology. We then ran a nonclock and a noncalibrated strict-clock analysis on a fixed topology (obtained in the previous analysis), allowing the priors' calculations. All posttree reconstruction analyses were conducted on the combined phylogenetic hypothesis obtained in BI with a model with two partitions.

ACOUSTIC RECORDINGS AND SOUND ANALYSIS

Calling males were located acoustically and visually at night from 19:00 to 24:00 h or during the day from 7:00 to 17:00 h for the diurnal species of the genus Staurois. Advertisement calls were recorded from a distance between 0.5 and 2 m. During each recording, a dim-red headlamp was used, in order to minimize any disturbance by light [START_REF] Pearse | The reactions of amphibians to light[END_REF][START_REF] Buchanan | Effects of enhanced lighting on the behaviour of nocturnal frogs[END_REF][START_REF] Koskelainen | Spectral sensitivities of short-and long-wavelength sensitive cone mechanisms in the frog retina[END_REF]. We used a Marantz PMD671 recorder with a Sennheiser ME64 microphone or an Avisoft CM16 microphone; sampling rate was 44.1 or 96 kHz at 16 bits. The call type most often heard from single males was considered as the advertisement call. Other call types produced by conspecific males, often heard when males were close to each other or physically engaged, were considered as aggressive or release calls, and they were disregarded. In order to complete our dataset, especially for taxa from nonsampled regions, recordings of 38 species were provided by collaborators or extracted from published compact discs (Table S3). Calls recorded for this study were deposited at the Fonoteca Zoológica in Madrid [START_REF] Toledo | The value of audiovisual archives[END_REF].

Approximately 21,500 notes (sensu [START_REF] Robillard | Evolution of advertisement signals in North American hylid frogs: vocalizations as endproducts of calling behavior[END_REF]) from 225 individuals in 79 species (from one to 14 individuals per species) were analyzed (Table S4). The sound files (WAV format) were labeled and segmented with custom-made scripts in the software Praat [START_REF] Boersma | Praat, a system for doing phonetics by computer[END_REF]. The segmented WAV files and the text file containing the labels and time information were then imported into the R environment (R Core Team 2013) and analyzed with the package seewave [START_REF] Sueur | Seewave, a free modular tool for sound analysis and synthesis[END_REF].

We selected acoustic variables according to two criteria: their relevance to our question, based on existing literature, and their availability for a large sample of species. Habitat acoustic properties are expected to impact the duration [START_REF] Morton | Ecological sources of selection on avian sounds[END_REF][START_REF] Dubois | A case of possible vocal convergence between frogs and a bird in Himalayan torrents[END_REF][START_REF] Ey | The "acoustic adaptation hypothesis" -a review of the evidence from birds, anurans and mammals[END_REF], redundancy [START_REF] Endler | Signals, signal conditions, and the direction of evolution[END_REF][START_REF] Ryan | Selection on long-distance acoustic signals[END_REF], dominant frequency (DF; [START_REF] Morton | Ecological sources of selection on avian sounds[END_REF][START_REF] Marten | Sound transmission and its significance for animal vocalization[END_REF]Arch and Narins 2008;Boonman and Kurniati 2011), bandwidth (BW;[START_REF] Dubois | A case of possible vocal convergence between frogs and a bird in Himalayan torrents[END_REF], and amplitude and frequency modulation (FM; [START_REF] Wiley | Physical constraints on acoustic communication in the atmosphere: implications for the evolution of animal vocalizations[END_REF][START_REF] Richards | Reverberations and amplitude fluctuations in the propagation of sound in a forest: implications for animal communication[END_REF] of the acoustic signal.

We therefore measured the following values from each note and averaged them for each individual and species for subsequent analyses: note duration, number of notes per call (defined here as an uninterrupted sequence of notes), DF, amplitude and FM within notes, and DF BW. For the amplitude and FM, the root-mean-square (RMS) sound pressure (in Pascal) and DF, respectively, were calculated for each 1-msec segment of the note. The slope of the resulting curve (RMS or DF values by time) was then calculated through linear model fitting. Each resulting curve was checked by eye. The values used for amplitude and DF modulations are thus representing slopes (without unit). This representation is more meaningful than amplitude or DF difference because it conveys information about the duration of the modulation: for example, a FM of 1 kHz would probably not have the same behavioral and evolutionary meaning over a 300-msecor a 3-sec-long note. [START_REF] Dubois | A case of possible vocal convergence between frogs and a bird in Himalayan torrents[END_REF] suggested that torrent-dwelling species produce short sequences of notes separated by long periods of silence and that the notes are rhythmically separated within the sequences. However, due to the high diversity in call structure in our dataset (including calls composed of a single note, one or several groups of notes, pulsed or nonpulsed notes), a comparative analysis of silence intervals based on the homology criterion was difficult to establish, and we limited our temporal analysis to the note duration and the number of notes per call.

CALLING SITE CHARACTERISTICS

In order to produce calling site categories, we measured habitat descriptors showed to be of importance in anuran habitat and calling site choice [START_REF] Gillespie | Herpetological richness and community structure of offshore islands of Sulawesi, Indonesia[END_REF][START_REF] Keller | The importance of environmental heterogeneity for species diversity and assemblage structure in Bornean stream frogs[END_REF][START_REF] Goutte | The importance of ambient sound level to characterise anuran habitat[END_REF]. Once an individual had been captured, we measured five habitat descriptors at the exact individual calling location: depth, width, and average slope of the closest water body; air temperature; canopy coverage; and ambient sound pressure level (SPL). In brief, ambient air temperature was measured with a probe-K digital thermometer (Hanna) to the nearest 0.1°C. The canopy coverage was measured the day following the capture with a convex-A spherical densiometer. The ambient SPL, in decibels, was measured with a digital SPL meter (American Recorder Technologies), with A-weighting and slow capture (1-sec averaging) to the nearest 0.1 dB (precision: 1.5 dB at 1 kHz). The maximal value for a 30-sec time window was taken. For ponds, we measured maximal depth and mean diameter. For streams, we measured the width of the water body, and the maximum depth across that width, at the focal male level. We also measured the maximal SPL in a 2-m radius around the frog calling post. A detailed description of habitat methods has been presented in an earlier paper [START_REF] Goutte | The importance of ambient sound level to characterise anuran habitat[END_REF]. We produced a broad-type coding for calling sites based on these precise measurements with multifactorial analysis and cluster analysis [START_REF] Goutte | The importance of ambient sound level to characterise anuran habitat[END_REF]: "torrent," "pond," and "river/lake." In the present study, we added the category "forest," as additional nonriparian species were included in this dataset.

For the fine-scale coding, we used the SPL and canopy coverage measures, two environmental variables predicted to impact call evolution [START_REF] Morton | Ecological sources of selection on avian sounds[END_REF][START_REF] Marten | Sound transmission and its significance for animal vocalization[END_REF][START_REF] Wiley | Physical constraints on acoustic communication in the atmosphere: implications for the evolution of animal vocalizations[END_REF][START_REF] Brumm | Acoustic communication in noise[END_REF][START_REF] Boonman | Evolution of high-frequency communication in frogs[END_REF][START_REF] Vargas-Salinas | Abiotic noise, call frequency and stream-breeding anuran assemblages[END_REF]. SPL was shown to be of the utmost importance to characterize frog habitats [START_REF] Goutte | The importance of ambient sound level to characterise anuran habitat[END_REF], whereas canopy coverage measures the openness of a site more precisely than the typically used binary character (i.e., "open" vs. "closed").

ANCESTRAL STATE ESTIMATION OF CALLING SITE TYPE AND CHARACTERISTICS

The choice of calling site may be subject to selection, as females may not mate with a male calling from a site unsuitable for egg laying, for example. Calling site preference can thus be considered as inheritable, even though, as any behavioral trait, it is also subjected to circumstantial factors [START_REF] Losos | Convergence, adaptation, and constraint[END_REF]. We reconstructed ancestral preference of calling sites using two coding options: broad-type and fine-scale. We reconstructed ancestral states of broad-type using the function rerootingMethod in the R package phytools [START_REF] Revell | phytools: an R package for phylogenetic comparative biology (and other things)[END_REF], and estimated the likelihood of each ancestral state with a Markov model allowing every transition rate between two states to be different [START_REF] Pagel | Detecting correlated evolution on phylogenies: a general method for the comparative analysis of discrete characters[END_REF][START_REF] Paradis | APE: analyses of phylogenetics and evolution in R language[END_REF]. For the fine-scale coding method, we estimated ancestral values of canopy coverage and SPL using the function fastAnc in the R package phytools, assuming a Brownian motion (BM) process. Before the analysis, we log-transformed the continuous variables to satisfy a normal distribution of the data. For both coding methods, the analyses were done on 500 trees, randomly sampled from the best-fitting trees in the BI analysis, to account for phylogenetic uncertainty. For each internal node of interest, we checked the monophyly across all sampled trees and excluded the trees for which the corresponding clade was nonmonophyletic.

RELATIONSHIP BETWEEN BODY SIZE AND CALL

CHARACTERISTICS

In order to assess whether morphological constraints on acoustic characters could impede our interpretation of the effect of environmental constraints on call evolution, we tested the relationship between body size and acoustic traits using phylogenetic generalized least squares (pGLS), with a correlation structure of the residuals corresponding to the phylogeny topology and chosen model of evolution (BM or Ornstein-Uhlenbeck [OU], see "Models of evolution" methods section below) for the given variable.

EVOLUTION OF CALL CHARACTERISTICS

Models of evolution

In the case of strong environmental pressures, similar environments may lead to similar phenotypes [START_REF] Williams | The origin of faunas. Evolution of lizard congeners in a complex island fauna: a trial analysis[END_REF][START_REF] Morton | Ecological sources of selection on avian sounds[END_REF][START_REF] Rothstein | Vocal dialects and their possible relation to honest status signalling in the brown-headed cowbird[END_REF]. Torrent frogs could thus display similar advertisement calls regardless of phylogenetic relatedness. To test this prediction, we fitted and compared models of evolution to our data. The BM model (Felsenstein 1985b), commonly used for trait evolution analyses, assumes that continuous characters can diverge indefinitely toward any value, which is generally not realistic for biological characters. The OU model corresponds to a BM process model with a stabilizing selection toward an optimum, denoted θ [START_REF] Hansen | Translating between microevolutionary process and macroevolutionary patterns: the correlation structure of interspecific data[END_REF]. The values of the traits are thus constrained around a given value and the strength of this constraint is controlled by another parameter, denoted α. An α-value of 0.001 reflects a very weak constraint of the trait toward the optimum θ, whereas an α of 100 corresponds to instantaneous transition to θ; if α equals 0, the OU model is reduced to a BM model. The parameter θ can be estimated for the whole phylogeny, or different values of θ can be estimated with changes in θ at nodes corresponding to a change in selective regime [START_REF] Hansen | Stabilizing selection and the comparative analysis of adaptation[END_REF][START_REF] Butler | Phylogenetic comparative analysis: a modeling approach for adaptive evolution[END_REF].

For each acoustic trait, we tested a series of models of evolution: a BM model assuming no trend or constraint (null model), an OU model assuming a single optimum θ, and five OU models assuming two to three optima in the phylogeny (Fig. 1). Models with multiple optima were defined according to biological hypotheses derived from the ancestral values estimation of calling site characteristics and previous hypotheses linking call features and environmental conditions [START_REF] Morton | Ecological sources of selection on avian sounds[END_REF][START_REF] Ryan | Selection on long-distance acoustic signals[END_REF][START_REF] Ey | The "acoustic adaptation hypothesis" -a review of the evidence from birds, anurans and mammals[END_REF][START_REF] Boonman | Evolution of high-frequency communication in frogs[END_REF]. They assume that extant taxa and ancestors belonging to the same "regime" are under the same evolutionary constraints. For each regime, a different θ was estimated. We also estimated α and the variance of the process (σ 2 ) for the whole phylogeny. As body size is often correlated to acoustic properties of vocalizations (e.g., [START_REF] Zimmerman | A comparison of structural features of calls of open and forest Habitat frog species in the central Amazon[END_REF][START_REF] Ryan | Selection on long-distance acoustic signals[END_REF][START_REF] Hoskin | Ecology and taxonomydriven deviations in the frog call-body size relationship across the diverse Australian frog fauna[END_REF][START_REF] Gingras | Call acoustics reflect body size across four clades of anurans[END_REF], and in order to test whether calling sites might have constrained the evolution of body size, we also performed the same analyses with the species average body size (snout-vent length).

Model OU2 assumed two regimes based on the major changes in calling site category estimated by the ASR (Fig. 2), one for torrent dwelling frogs (Staurois, CHM, Amolops and Odorrana) and another one for the other frogs. Model OU2b assumed the same groups, except that the lineage Amolops and non-torrentdwelling species shared the same regime. This model was based on the ancestral values estimation of precise calling site characteristics (SPL and canopy coverage, Fig. 3), which showed similar values for the lineage Amolops and non-torrent-dweller groups. Model OU3 assumed three regimes and three major changes in calling site: a first change toward torrent life at node 5 (Fig. 2), placing the groups Staurois, CHM, and Amolops in the same regime; a second change after the divergence of Amolops (node 13, Fig. 2); and a third change before the divergence of Odorrana (node 21, Fig. 2). In this model, the lineage Odorrana is assumed to have a different regime than the other torrentdwelling groups because it corresponds to a different evolutionary event, potentially involving different mechanisms. All nontorrent-dwelling groups shared a single regime. Model OU3b, as for model OU2b, only departed from the previous model regarding the placement of the lineage Amolops in the nontorrent regime. Model OU3c is based on call similarities observed in our dataset: it assumed a convergence between torrent-dwelling groups, Amolops and Staurois on one hand, which shared the same regime, and Odorrana and CHM on the other hand, which shared another regime. The non-torrent-dwelling lineages shared a third regime.

We chose to keep both α and σ 2 constant across the whole phylogeny to avoid overparameterization; preliminary analyses showed that a larger dataset would be needed to test more complex models where one or both of these parameters could vary among regimes while keeping enough statistical power (data not shown). We tested whether torrent-dwelling groups had optimal trait values different from those of nontorrent species (models BM vs. OU2 and OU1 vs. OU2), and if they all shared the same optimal trait values, or whether they exhibited different "strategies" evolving toward different optimal values (models OU2 vs. OU3 and OU3 vs. OU3c). Finally, we tested whether the torrent lineage Amolops shared optimal trait values with non-torrent-dwelling species (models OU2 vs. OU2b and OU3 vs. OU3b).

In order to have a robust selection of models of evolution, we followed the method described in [START_REF] Boettiger | Is your phylogeny informative? Measuring the power of comparative methods[END_REF] simulating 1000 datasets for each model to take into account phylogenetic uncertainty and variance in parameter estimations. We first estimated the parameters of the chosen models from the data with the function OUwie, in the R package OUwie [START_REF] Beaulieu | Modeling stabilizing selection: expanding the Ornstein-Uhlenbeck model[END_REF][START_REF] Beaulieu | OUwie: analysis of evolutionary rates in an OU framework[END_REF]. We then simulated the data a hundred times with the estimated parameters under each model using the function rTraitCont in the R package ape [START_REF] Paradis | APE: analyses of phylogenetics and evolution in R language[END_REF][START_REF] Popescu | ape 3.0: New tools for distance-based phylogenetics and evolutionary analysis in R[END_REF] and fitted both models again on the simulated data. For each simulated dataset, the likelihood ratio between the two models was calculated. To choose the best-fitting model, we finally compared the distribution of likelihood ratios for the simulations under both models to the likelihood ratio derived from the real data. We repeated this protocol on 10 post-burnin trees from the BI analysis. The pairwise models comparisons were chosen in a stepwise manner, comparing increasingly complex models (Fig. 1). This method allows the assessment of the power of our S5. Torrent-dwelling clades present higher canopy coverage and louder ambient noise level than non-torrent-dwelling clades with a few exceptions, in particular the genus Amolops.

analysis simultaneously to the models comparison. In contrast, AIC scores (Table S9), although allowing the comparison of all models at the same time, can be misleading in favoring more complex but poorly supported models [START_REF] Boettiger | Is your phylogeny informative? Measuring the power of comparative methods[END_REF].

Ancestral values' estimation of vocalization characteristics and body size

We estimated ancestral values of vocalization characteristics and body size with the function fastAnc in phytools [START_REF] Revell | phytools: an R package for phylogenetic comparative biology (and other things)[END_REF]) on a set of 500 trees. Again, each variable was log-transformed, except for body size, from which the square root was taken prior to the analysis. According to the chosen model of evolution (see above), we used either a BM or an OU model of evolution to compute these estimates. In the latter case, we transformed the branch lengths of the tree using the fitted value of α with the function ouTree (R package geiger) before estimating the ancestral values using BM, thereby obtaining ancestral values under the OU model.

Results

MOLECULAR PHYLOGENY

The resulting topology in BI (Fig. S1) agrees with the most recent published phylogeny of the family Ranidae [START_REF] Pyron | A large-scale phylogeny of Amphibia including over 2800 species, and a revised classification of extant frogs, salamanders, and caecilians[END_REF]. The few differences (A. bellulus, Rana weiningensis, R.

shuchinae, Glandirana minima, Pelophylax saharicus) were not strongly supported in one or both of the phylogenies (low support values or position method-dependent).

The phylogenetic reconstruction in ML (Fig. S2) resulted in a very similar topology. Differences affected recent nodes, which were not or weakly supported in one or both topologies. These differences have a minimal impact on our conclusions about vocalization evolution as they concern only recent nodes within clades sharing similar environmental constraints and vocalization characteristics.

TAXONOMIC NOTE

In this article, we follow [START_REF] Frost | Amphibian species of the world: an online reference[END_REF] taxonomy. As seen in previously published phylogenies [START_REF] Pyron | A large-scale phylogeny of Amphibia including over 2800 species, and a revised classification of extant frogs, salamanders, and caecilians[END_REF], the genus Huia is not monophyletic in our phylogenetic hypothesis, placing the clade containing "Huia" masonii and "Huia" sumatrana as sister taxa to the genus Clinotarsus (Fig. S1). As Huia cavitympanum is the type species of the genus, a taxonomic revision is required for this clade.

The genus Glandirana, as defined in [START_REF] Frost | Amphibian species of the world: an online reference[END_REF], is not monophyletic in our phylogenetic hypotheses, although the problematic position of Glandirana minima is not supported in any tree. Here, we follow [START_REF] Fei | Fauna sinica: amphibia. Vol. 3 Anura Ranidae[END_REF] and recognize Glandirana minima, Rugosa emeljanovi, and R. tientiaensis.

EVOLUTION OF CALLING SITE PREFERENCES AND INFLUENCE OF CODING OPTIONS

The estimation of ancestral calling site values using either the broad-type or the fine-scale coding methods leads to different phylogenetic patterns. Broad-type allowed splitting the phylogeny into clearly defined groups, whereas fine-scale descriptors varied in more complex ways.

The broad-type approach suggests that the ancestor of the Ranidae (sensu [START_REF] Frost | The amphibian tree of life[END_REF] reproduced in fast-flowing streams (Fig. 2, Table S5). Our ancestral reconstruction of calling site for nodes deeper than the Ranidae ancestor may be strongly constrained by the outgroup sampling, which is limited by definition. It is thus difficult to say whether the common ancestor of the Ranidae and Rhacophoridae was already a torrent dweller or whether the Ranidae ancestor pioneered this habitat. The first diverging groups (i.e., Staurois, Clinotarsus, Huia, Meristogenys, and Amolops) remained in torrents for reproduction. At node 13, we inferred a change in calling site preference toward calmer water bodies. Indeed, species of the genera Pelophylax, Sanguirana, Glandirana, Rugosa, Hylarana, Lithobates, and Rana are found calling at ponds, lakes, rice fields, or calmer riverbanks. We inferred a secondary change toward faster-flowing streams at the divergence of the genus Odorrana.

Ancestral estimation of the fine-scale characteristics provides a more-detailed scenario on the evolution of calling site prefer-ences and showed clear variations within the "torrent" category. The patterns of variation of ambient noise level (SPL) and canopy coverage showed some clear similarities (Fig. 3, Table S5). These variables are related to one another, and it is thus not surprising to see a correlation in their variation patterns. Both variables had relatively high values for the ancestor of Ranidae. They decreased gradually through evolutionary time, but increased twice after the divergences of Staurois and the Clinotarsus-Huia-Meristogenys (CHM) group.

The canopy coverage increased dramatically again after the divergence of Odorrana, whereas the increase in ambient noise level occurred only after the divergence of a subgroup of Odorrana. The values of both variables varied much less after the divergence of Amolops. We observed a decreasing trend for both variables for the nontorrent groups.

RELATIONSHIP BETWEEN BODY SIZE AND CALL

CHARACTERISTICS

Body size was significantly correlated with DF, note duration, and AM (Fig. 4, Table S8). There was no significant correlation with frequency BW, FM, or the number of notes per call. A large majority of torrent-dwelling species had a higher DF than expected from their body size, based on all sampled ranids (Fig. 4), although they did not deviate from other species for all other acoustic variables.

EVOLUTION OF CALLS AND BODY SIZE

Models of evolution

For all acoustic variables except the number of notes per call, the best-fitting model separated the torrent-dwelling ranids from the non-torrent-dwelling groups (Table 1), each group showing different optima (Table 2). The OU and BM models for the number of notes per call were not significantly different. For the FM, the model OU2b was preferred over the model OU2 in nine of the ten trees sampled (Table 1), even though the power of the test remained low (52.8%). For body size, no OU model was significantly better than the BM model (Table 1).

The optimal values (θ) estimated for torrent-dwelling ranids were a higher DF (7.7 vs. 2.0 kHz), a shorter note duration (56 vs. 99 msEC), and a greater FM (threefold increase) than the optima for non-torrent-dwelling lineages (Table 2). The optima for AM were not significantly different between the torrent-and non-torrent-dwelling lineages.

For DF, the estimated value for α was low (Table 2), indicating a weak constraint the estimated optimal value θ; this constraint was more substantial for the FM and note duration; and the estimated values of α for BW and AM were very high, indicating a strong selective pressure for this trait. As α was estimated for the whole phylogeny and not for each regime, its value does not account for the possibility that some regimes may involve a stronger constraint than others. We will thus refrain from discussing it further.

Ancestral values' estimation

According to our ancestral value estimations (Table 3), the ancestor of the Ranidae (node 5) displayed a multinote call, with a DF high enough to be placed in the torrent-dwelling frogs partition (2.9 kHz). Four increases of DF, linked to the four torrentdwelling lineages, were estimated: in the Staurois lineage (node 6), in the CHM group (node 9), after the divergence of Odorrana (node 21), and a slighter one in the genus Amolops (node 12). On deeper nodes, the DF value decreased gradually from the Ranidae ancestor to the Odorrana divergence.

Note duration decreased for the genus Staurois. Conversely, note duration increased for the non-torrent-dwelling group nodes, except for the genus Babina (node 20). Note duration decreased again after the divergence of Odorrana (node 21).

The number of notes per call increased after the divergence of Staurois and Amolops, and decreased after the divergence of the CHM group and Odorrana. The frequency BM increased after the divergence of the CHM group and Odorrana, but decreased after the divergence of the genus Staurois. FM increased after the divergence of the CHM group and Odorrana, but had low values at nodes basal to Amolops and non-torrent-dwelling groups, except for the genus Babina. Finally, AM showed little variation through the phylogeny. Body size showed an overall increase throughout the phylogeny but no clear pattern emerged (Fig. S3).

Model choice for the call characteristics and body

Discussion

In this study, we tested whether similarly constraining environments would lead to similar vocalizations as predicted by the AAH [START_REF] Morton | Ecological sources of selection on avian sounds[END_REF][START_REF] Ey | The "acoustic adaptation hypothesis" -a review of the evidence from birds, anurans and mammals[END_REF]. We showed that (1) the level of details in habitat characterization impacts the interpretations on environmental constraints and (2) these constraints constitute an important factor of call evolution in torrent-dwelling ranids.

EVOLUTION OF CALLING SITE PREFERENCE AND INFLUENCE OF CODING METHODS

Despite straightforward expectations, studies on the AAH have produced contradictory and inconclusive results [START_REF] Ey | The "acoustic adaptation hypothesis" -a review of the evidence from birds, anurans and mammals[END_REF]. This discrepancy might suggest that environmental constraints are not a major selective force on calls or, as suggested lately [START_REF] Bosch | Are frog calls modulated by the environment? An analysis with anuran species from Bolivia[END_REF][START_REF] Ey | The "acoustic adaptation hypothesis" -a review of the evidence from birds, anurans and mammals[END_REF], that environmental constraints are not suitably characterized to demonstrate their influence. In the AAH framework, the environment is most often broadly defined as "open" or "closed," which is likely too coarse a definition to account for the microscale habitat variability. Given that any eco-evolutionary pattern is scale-dependent [START_REF] Levin | The problem of pattern and scale in ecology: the Robert H. MacArthur award lecture[END_REF]), a broad definition process might lead to spurious conclusions, as already noted in ecology and ethology [START_REF] Robillard | Evolution of advertisement signals in North American hylid frogs: vocalizations as endproducts of calling behavior[END_REF][START_REF] Grandcolas | Mapping extrinsic traits such as extinction risks or modelled bioclimatic niches on phylogenies: does it make sense at all?[END_REF][START_REF] Goutte | The importance of ambient sound level to characterise anuran habitat[END_REF][START_REF] Legendre | Dyadic behavioural interactions in cockroaches (Blaberidae): ecomorphological and evolutionary implications[END_REF].

Contrasting two coding options for calling sites, we show here that a fine-scale approach is needed. First, even though both coding methods are based on precise measurements (the broadtype were defined using precise measurements-see [START_REF] Goutte | The importance of ambient sound level to characterise anuran habitat[END_REF] for details), they suggest different scenarios for the lineage Amolops: grouped with torrent-dwelling lineages with "broad" habitat categories, or grouped with non-torrent-dwelling lineages for the fine-scale approach. This implies diverging appraisals on environmental constraints, and consequently on convergence and adaptation [START_REF] Patterson | Homology in classical and molecular biology[END_REF][START_REF] Wake | Homoplasy: the result of natural selection, or evidence of design limitations?[END_REF]. Second, according to the AAH, note duration should be longer in closed habitats than in open habitats, seen as more fluctuant because of air turbulence, temperature gradients, and wind [START_REF] Morton | Ecological sources of selection on avian sounds[END_REF][START_REF] Ey | The "acoustic adaptation hypothesis" -a review of the evidence from birds, anurans and mammals[END_REF]. Yet, in our dataset, note duration decreases with increasing canopy coverage. We argue, however, that this result does not necessarily contradict the AAH predictions: even in a dense forest, a torrent is acoustically different from a closed habitat as described in the AAH literature. We can expect the atmosphere around a torrent to be highly turbulent, because a torrent constitutes a break through the vegetation allowing the wind in, while cold turbulent water creates temperature gradients. Thus, torrents fit neither with the "open" nor with the "closed" categories, but present some constraints similar to both habitats. The AAH prediction involves air turbulence, wind, and temperature gradients, three variables that should be measured to properly test the aforementioned prediction. In other words, detailed habitat characterization should always be preferred to investigate the AAH. A few studies followed this direction but only at a small taxonomic scale so far [START_REF] Castellano | Call degradation in diploid and tetraploid green toads[END_REF][START_REF] Ziegler | Linking amphibian call structure to the environment: the interplay between phenotypic flexibility and individual attributes[END_REF]. Framed in a large phylogenetic scale and using fine-scale environmental data, our study shows that several acoustic features (DF, note duration, BW frequency, and DF modulation) are compatible with the AAH predictions (see below).

CALL EVOLUTION, ENVIRONMENTAL AND MORPHOLOGICAL CONSTRAINTS

The best-fitting model of evolution for all but one vocalization characteristic distinguishes torrent versus non-torrent-dwelling groups as being under different evolutionary constraints despite the existent morphological constraint on some vocalization characteristics (Fig. 4). Although our data did not allow the distinction of different selective regimes among the different torrent dwelling groups, the ancestral state reconstruction of both calling site and call characteristics and the AIC scores of the models of evolution (Table S9) suggest two distinct call types among torrent-dwelling ranids. More complex models, involving several strategies for torrent-dwelling groups or allowing different alpha parameters for each regime, should be investigated with a larger dataset in order to test them with sufficient power.

In still-water bodies, male vocalizations are relaxed from the torrent-related selective pressures, so that other factors, such as female choice, competition, or predator-based selection may drive the evolution of vocalizations [START_REF] Ryan | Selection on long-distance acoustic signals[END_REF]. This could have resulted in the diversity of calls we observed for nontorrent-dwelling species in our data (see Table S4). Torrents are seen as constraining environments regarding acoustic communication [START_REF] Feng | Ultrasonic communication in frogs[END_REF], so that convergences and adaptations are expected [START_REF] Trontelj | Ecomorphological convergence of cave communities[END_REF][START_REF] Weir | The role of ecological constraint in driving the evolution of avian song frequency across a latitudinal gradient[END_REF][START_REF] Stayton | The definition, recognition, and interpretation of convergent evolution, and two new measures for quantifying and assessing the significance of convergence[END_REF].

For torrent-dwelling ranids, we inferred a maximum of two calling "strategies," divided into two nonmonophyletic groups: Staurois-Amolops and CHM-Odorrana. This low diversity in call types supports the hypothesis that torrents environmental properties have channeled the evolution of ranid vocalizations, like environmental factors constrain morphological or other behavioral traits [START_REF] Trontelj | Ecomorphological convergence of cave communities[END_REF][START_REF] Roda | Convergence and divergence during the adaptation to similar environments by an Australian groundsel[END_REF]. The similarities in vocalizations between Odorrana and CHM species fit with the definition of a convergence [START_REF] Stayton | The definition, recognition, and interpretation of convergent evolution, and two new measures for quantifying and assessing the significance of convergence[END_REF]. Nevertheless, our dataset was insufficient to distinguish between the model of evolution supporting this convergence hypothesis (OU3c) and the one rejecting it (OU3), even though the AIC scores favored the OU3c model for three acoustic characteristics (DF, frequency BW, and number of notes per call). The call characteristics of Staurois and Amolops are more similar to the estimates of the Ranidae ancestor vocalization. Staurois and Amolops exhibit retained similarities, inherited from their common ancestor, and not a convergence pattern [START_REF] Stayton | The definition, recognition, and interpretation of convergent evolution, and two new measures for quantifying and assessing the significance of convergence[END_REF]. In torrent ranids, two major options in advertisement calls evolved, one inherited from the Ranidae ancestor and the other appearing convergently in two ranid groups. These two options underline that different solutions might evolve despite similar constraints, which is rarely supposed at a high level of organization [START_REF] Edelman | Degeneracy and complexity in biological systems[END_REF][START_REF] Roda | Convergence and divergence during the adaptation to similar environments by an Australian groundsel[END_REF] or that subtle differences among the constraints resulted in different solutions. For instance, the group CHM might have evolved a different calling "strategy" than their closely related torrent-dwelling lineages (i.e., Staurois and Amolops) after a change in calling site characteristics toward larger streams (Table 3). Indeed, the vocalizations of the former are composed of single or very few, modulated notes and present high frequencies up to ultrasonic frequencies, while the latter relatively low-pitch calls are composed of multiple short notes. Although the AIC scores of the models support this hypothesis and the fine-scale coding approach already brings substantial elements, more data are needed to confirm this hypothesis with sufficient power.

The evolution of call DF follows patterns of ambient noise level variation (Fig. 3 and Table 3), indicating a possible adaptation of the former to the latter: for the lineage Amolops both the ambient noise level and the DF increased marginally, whereas both values increased substantially for the other torrent-dwelling groups. The adaptive value of higher-pitched vocalizations in noisy environments has been proposed as an advantageous masking release mechanism for birds [START_REF] Slabbekoorn | Birds sing at a higher pitch in urban noise[END_REF][START_REF] Narins | Old World frog and bird, vocalizations contain prominent ultrasonic harmonics[END_REF][START_REF] Brumm | Acoustic communication in noise[END_REF][START_REF] Nemeth | Blackbirds sing higher-pitched songs in cities: adaptation to habitat acoustics or side-effect of urbanization?[END_REF][START_REF] Cardoso | On the relation between loudness and the increased song frequency of urban birds[END_REF][START_REF] Goodwin | Shift of song frequencies in response to masking tones[END_REF] and anurans [START_REF] Narins | Old World frog and bird, vocalizations contain prominent ultrasonic harmonics[END_REF]Arch and Narins 2008;Arch et al. 2008;[START_REF] Cunnington | Plasticity in the vocalizations of anurans in response to traffic noise[END_REF][START_REF] Boonman | Evolution of high-frequency communication in frogs[END_REF]. Our results support this hypothesis although comparative and experimental approaches are needed to further support these findings. The increase of call DF in Staurois is, however, minor compared with the other groups, even though the individuals from this genus vocalize at sites with the highest noise level.

Three other vocalization characteristics show general evolutionary patterns related to SPL: note duration decreases, frequency BW, and FM increase at nodes showing an increase in SPL (Fig. 3 and Table 3). However, for BW and FM, the genus Staurois departs from the general trend once again. These differences may be explained by the visual displays these species use during advertisement in addition to vocalizations [START_REF] Grafe | Multimodal signaling in male and female foot-flagging frogs Staurois guttatus (Ranidae): an alerting function of calling[END_REF][START_REF] Preininger | Communication in noisy environments II: visual signaling behavior of male foot-flagging frogs Staurois latopalmatus[END_REF][START_REF] Grafe | Multimodal communication in a noisy environment: a case study of the Bornean rock frog Staurois parvus[END_REF]. Visual communication could lower the selective pressure on the acoustic signal, which may then act more as an alerting call than an advertisement call per se [START_REF] Grafe | Multimodal signaling in male and female foot-flagging frogs Staurois guttatus (Ranidae): an alerting function of calling[END_REF]. Such bimodal communication is known only for Staurois within the Ranidae, preventing from any statistical test of this hypothesis. However, torrent-dwelling species from other families are known to use bimodal communication [START_REF] Lindquist | Field studies on visual and acoustic signaling in the "earless" Panamanian golden frog, Atelopus zeteki[END_REF][START_REF] Haddad | Visual and acoustic communication in the Brazilian torrent frog, Hylodes asper (Anura: Leptodactylidae)[END_REF][START_REF] Amézquita | How, when, and where to perform visual displays: the case of the Amazonian frog Hyla parviceps[END_REF][START_REF] Gomez | Costly help of audiovisual bimodality for female mate choice in a nocturnal anuran (Hyla arborea)[END_REF][START_REF] Preininger | Multimodal signaling in the Small Torrent Frog (Micrixalus saxicola) in a complex acoustic environment[END_REF]) and they should be investigated in this respect.

Although body size was significantly correlated with three vocalization characteristics (Fig. 4), calling site categories did not influence the evolution of body size (Table 1). The observed influence of calling site on the evolution of vocalization was therefore not mediated by a change in body size. Moreover, the quasi-totality of torrent-dwelling species had a higher DF than expected for their size based on the relationship observed in all sampled ranids (Fig. 4), indicating that selection of high DFs in torrent habitat occurred in spite of this morphological constraint.

Despite coarsely similar environmental conditions, we determined two calling "strategies" for torrent-dwelling ranids. This result shows that, while investigating the effect of environmental constraints on call evolution, a fine-scale characterization of the habitat, rather than broad categories, is needed. Comparative studies relying on fine-scale measures (environmental but also morphological and behavioral) would certainly improve our un-derstanding of the evolution of acoustic communication, and reduce the discrepancies between theoretical and empirical studies in the AAH framework.

Figure 1 .

 1 Figure 1. Tested OU models of evolution. Panels represent models from a single regime of selection (OU1) through multiple regimes derived from biological hypotheses (OU2-3c). Regimes are color-coded. θ is estimated for each regime while α and σ 2 are estimated for the whole phylogeny and kept constant.

Figure 2 .Figure 3 .

 23 Figure 2. Ancestral state reconstructions of habitat types. Histograms at numbered nodes indicate the probability of each habitat type for 500 trees. Probabilities under 0.2 are not plotted. Asterisks indicate a posterior probability of 1 for the corresponding nodes. Four torrent-dwelling clades are identified (Staurois, Huia-Clinotarsus-Meristogenys, Amolops, and Odorrana), with an ancestral torrent habitat for the family Ranidae. Calmer water bodies are inferred for nodes 13-18 so that torrent-dwelling life for Odorrana species corresponds to a reversion toward "torrents."

Figure 4 .

 4 Figure 4. Relationships between body size and call acoustic characteristics. Panels show relationships between body size (SVL, squarerooted) and call acoustic characteristics (log-transformed) through pGLS regression, with regression line in red and associated P-values. Red points: torrent-dwelling species; black points: non-torrent-dwelling species. Three call characteristics shows significant relationship to body size (top panels) and most torrent-dwelling species show a higher DF than expected from their body size.

  size based on 100 simulations for a set of 10 trees. The percentage of rejections of the first model is given (Rej.), with the average and 95% confidence interval of the associated power of the test (β). Abreviations for call characteristics: AM, amplitude modulation; DF, dominant frequency; BW, bandwidth; FM, frequency modulation.

Table 1 .

 1

Table 2 .

 2 Estimates of the OU2 model.

		AM	DF (Hz)	Frequency BW (Hz)	FM	Note duration (msec)
	θ pond	7.12 ± 1.86	2043.53 ± 3.94	963.36 ± 2.21	2.86 ± 2.18	99.11 ± 1.85
	θ torrent	6.72 ± 1.46	7712.52 ± 1.41	1478.17 ± 1.08	9.27 ± 1.63	56.15 ± 1.34
	α	232.25	9.22	325.33	36.45	33.08
	σ 2	1432.71	6.61	92.19	293.67	71.88
	θ

estimates have been back-transformed and expressed in the original variable units with standard error. Modulation measures have no unit. Abreviations for call characteristics: AM, amplitude modulation; DF, dominant frequency; BW, bandwidth; FM, frequency modulation. Table 3. Ancestral value estimations for vocalization characteristics.

				Frequency BW		Note duration
		AM	DF (Hz)	(Hz)	FM	(msec)	Notes per call
	1	7 . 0 4± 1.57	2593.10 ± 1.78	1132.69 ± 1.12	4.30 ± 1.90	79.37 ± 1.40	4.31 ± 6.66
	2	6.89 ± 1.54	2594.79 ± 1.67	1132.53 ± 1.11	4.32 ± 1.89	79.55 ± 1.39	4.86 ± 3.56
	3	7 . 0 4± 1.57	2630.94 ± 1.61	1132.69 ± 1.11	4.31 ± 1.89	79.38 ± 1.40	5.32 ± 2.74
	4	7.05 ± 1.57	2580.21 ± 1.57	1132.68 ± 1.11	4.32 ± 1.91	79.03 ± 1.41	6.32 ± 1.90
	5	7 . 0 3± 1.57	2895.48 ± 1.41	1132.99 ± 1.12	4.40 ± 1.86	80.91 ± 1.38	5.32 ± 2.74
	6	7.07 ± 1.82	3883.52 ± 1.64	1123.83 ± 1.15	8.88 ± 3.62	58.78 ± 1.94	7.55 ± 1.20
	7	7 . 2 6± 2.57	4239.53 ± 1.45	1045.92 ± 1.23	31.63 ± 4.88	31.82 ± 2.21	18.82 ± 0.70
	8	7.02 ± 1.57	2876.98 ± 1.38	1133.07 ± 1.12	4.39 ± 1.86	81.36 ± 1.38	5.47 ± 0.90
	9	7 . 0 3± 1.59	4610.08 ± 1.56	1136.83 ± 1.12	4.85 ± 2.13	83.20 ± 1.50	3.11 ± 1.30
	10	7.12 ± 1.69	5612.64 ± 1.60	1155.26 ± 1.13	6.70 ± 2.92	80.82 ± 1.75	1.67 ± 1.19
	12	7.02 ± 1.58	2861. 00 ± 1.48	1133.12 ± 1.12	4.29 ± 1.95	80.30 ± 1.43	9.33 ± 1.16
	14	7.02 ± 1.57	2449.78 ± 1.39	1132.80 ± 1.12	4.39 ± 1.91	81.97 ± 1.40	4.79 ± 0.88
	16	6.95 ± 1.60	1974.88 ± 1.61	1131.94 ± 1.12	3.92 ± 2.25	90.13 ± 1.54	4.97 ± 1.43
	17	6.96 ± 1.58	2056.07 ± 1.43	1132.98 ± 1.12	4.20 ± 1.96	85.91 ± 1.42	4.53 ± 0.98
	18	6.77 ± 1.62	1323.19 ± 1.57	1128.94 ± 1.12	3.52 ± 2.36	103.03 ± 1.58	5.14 ± 1.21
	19	6.95 ± 1.59	2168.58 ± 1.49	1134.16 ± 1.12	4.35 ± 2.09	83.60 ± 1.48	4.53 ± 0.98
	20	6.72 ± 2.43	1265.28 ± 1.47	1115.70 ± 1.22	6.46 ± 4.82	77.13 ± 2.20	6.42 ± 0.76
	21	6.89 ± 1.65	2875.54 ± 1.52	1142.92 ± 1.13	4.85 ± 2.56	75.47 ± 1.64	1.93 ± 1.06
	22	6.85 ± 1.68	3146.11 ± 1.53	1147.02 ± 1.13	5.23 ± 2.75	73.29 ± 1.69	1.93 ± 1.06
	23	6.

41 ± 1.07 4627.40 ± 1.49 1210.06 ± 1.18 9.92 ± 4.11 60.99 ± 2.04 1.28 ± 0.83 Mean and 95% confidence interval are given. Ancestral values for nodes corresponding to torrent-dwelling species are in bold. Estimations were obtained with an OU model, except for the number of notes per call (BM model). Abreviations for call characteristics: AM, amplitude modulation; DF, dominant frequency; BW, bandwidth; FM, frequency modulation.

ACKNOWLEDGMENTS

We are very thankful to D. Bickford, W. Bin, U. Grafe, D. Iskandar, J. Jianping, W. Jichao, L. Maklarin, A. Ohler, and P. Yambun who helped in the field and with the permits; to A. Boonman and D. Preininger who shared their recordings; and to F. Moreau who helped with the molecular work. We are very thankful to W. Hödl, C. Miaud, E. Paradis, and A. Pyron for their comments on earlier versions of the manuscript. We also thank the Fonoteca Zoológica (www.FonoZoo.com), Museo Nacional de Ciencias Naturales -CSIC Madrid, for providing frog recordings. Molecular phylogenetic reconstructions were done with the cluster of UMS 2700 CNRS MNHN, Paris, and CIPRES online tools (Miller et al. 2010).

This project was supported by the network "Bibliothèque du Vivant" funded by the, the Muséum National d'Histoire Naturelle (MNHN), the INRA and the CEA (Centre National de Séquenc ¸age), internal grants (ATM "Biodiversité fossile et actuelle" supported by P. Janvier and S. Peigné) from MNHN, the École Normale Supérieure de Lyon, the National University of Singapore, the Société des Amis du Muséum, the Ministerio de Ciencia e Innovación, Spain (projects TATANKA CGL2011-25062, CGL2010-09700), and the Ministerio de Economía y Competitividad, Spain (project CGL2011-16159-E; PI. R. Márquez).

DATA ARCHIVING

Original DNA sequences are archived in GenBank database (accession numbers KU840478-KU840796). Calls recorded for this study were de-

Supporting Information

Additional Supporting Information may be found in the online version of this article at the publisher's website: S1. Primers. Table S2. GenBank accession numbers. Table S3. List of species recordings provided by recorders other than S. Goutte. Table S4. Call variables' average values per species. Table S5. Ancestral state reconstructions for the calling site types and the habitat variables. Table S6. Description of the biological hypotheses for each Ornstein-Uhlenbeck (OU) model used in the analysis. Table S7. Ancestral value estimation for body size (SVL). Table S8. Correlations between the body size and acoustic variables by phylogenetic least squares. Table S9. Fit of the models of evolution for the acoustic variables. Table S10. Parameter values for the models of evolution fitted to acoustic variables and body size.


Benjamin Le Delliou
email: benjamin.ledelliou@agroparistech.fr

Olivier Vitrac
email: olivier.vitrac@agroparistech.fr

Sandra Domenek
email: sandra.domenek@agroparistech.fr

BIOPOL-2019

Introduction

Materials with strong environmental impact are still used in disposable products such as food packaging materials. Alternative materials bio-sourced or biodegradable are highly desirable. Polyhydroxyalkanoates (PHAs), obtained from microbial fermentation, meet the two qualities by being bio-based and biodegradable but show a lack of properties showing a relatively high stiffness and brittleness while showing a relatively high glass transition temperature (0-5°C) compared to its counterpart low density polyethylene 2 . A conventional strategy to improve thermomechanical properties of the polymer consists in blending the brittle material with a ductile material. We report the results of blending PHBV with poly(butylene succinate-co-adipate) (PBSA) by melting state at different PHBV/PBSA content weight ratio. PBSA is a biodegradable aliphatic polyester, showing a low melting temperature, good thermal stability and low glass transition temperature (-45°C).

Materials & Methods

Commercial PHBV, under the trade name PHI 002 containing 3 mol% HV according to its technical data sheet and PBSA under the trade name PBE 001 were purchased from NaturePlast. The materials were dried at 70 °C for at least six hours, melt-blended in a mixing chamber of an internal mixer SCAMEX Rheoscam (France) at 190 °C for 9min at 90rpm and finally processed by thermal molding at 180 °C and 150 bars (SCAMEX 15T, France). The effect of PBSA content on the glass transition temperature (Tg) of PHBV was investigated by Dynamic Mechanical Analysis (DMA) using a TRITEC 2000 DMA (Triton Technology Ltd., UK)) by applying a temperature scan from -60 °C to 190 °C. The thermal stability of the blends was investigated by Thermo-Gravimetric Analysis (TGA, Q500, TA Instruments) in Hi-Res Mode. An Environmental Scanning Electronic Microscope (ESEM, FEI Quanta 200) was used to characterize the phase morphology. The samples were first cryo-fractured using liquid nitrogen and then sputter-coated with a thin gold layer (Sputter coater Emitech K550).

Results & Discussions

Figure 1 shows the evolution of the torque measured in the mixing chamber during blending. A large decrease in torque is observe when the PBSA is increased beyond 50wt%. This evolution is consistent with the decrease in viscosity of the binary blends. The drop of viscosity at a particular threshold was tested and showed that beyond 50wt% PBSA, melting properties of the binary mixtures in the mixing chamber were driven by PBSA. At high quantities of PBSA, an increase in torque can be observed over time, which could be due to a cross-linking between PHBV and PBSA.

Figure 1 :

 1 Figure 1: Measurement of torque over time during melt mixing of PHBV and PBSA Figure 2 shows the obtained phase morphology for the binary blend PHBV/PBSA 70/30 w/w and present nodular structures. While, DMA results presented in Figure 3 show the broadening of the Tg peak which suggests the presence of three phases, pure PHBV, pure PBSA and mixed PHBV/PBSA and correlates with the assumption of crosslinking made previously. The mixture has two Tg values with a value associated to PHBV phase decreased to 18.3 °C from 21.6 °C. PBSA increased the chain mobility of PHBV in the miscible region and hence conferring a toughening effect. Finally a significant improvement of thermomechanical properties of PHBV/PBSA blends comparatively to neat PHBV is obtained.

Figure 2 :Figure 3 :

 23 Figure 2: SEM images of cryo-fractured surfaces of PHBV/PBSA 70/30 w/w