Thomas De Solan|

Marc Théry

Damien Picard

Pierre-André Crochet

Patrice David

Jean Secondi

A lot of convergence, a bit of divergence: environment and interspecific interactions

Keywords: Reproductive character displacement, natural selection, sexual selection, crypsis, Lissotriton

Coexistence with related species poses evolutionary challenges to which populations may react in diverse ways. When exposed to similar environments, sympatric populations of two species may adopt similar phenotypic trait values. However, selection may also favor trait divergence as a way to reduce competition for resources or mates. The characteristics of external body parts, such as coloration and external morphology, are involved to varying degrees in intraspecific signaling as well as in the adaptation to the environment, and consequently may be diversely affected by interspecific interactions in sympatry. Here, we studied the effect of sympatry on various color and morphological traits in males and females of two related newt species Lissotriton helveticus and L. vulgaris. Importantly, we did not only estimate how raw trait differences between species respond to sympatry, but also the marginal responses after controlling for environmental variation. We found that dorsal and caudal coloration converged in sympatry, likely reflecting their role in adaptation to local environments, especially concealment from predators. In contrast, aspects of male and female ventral coloration, which harbours sexual signals in both species, diverged in sympatry. This divergence may reduce opportunities for interspecific sexual interactions and the associated loss of energy, suggesting reproductive character displacement (RCD). Our study emphasizes the contrasting patterns of traits involved in different functions and calls for the need to consider this diversity in evolutionary studies.

INTRODUCTION

Geographic patterns of convergence and divergence among species have attracted a lot of interest in evolutionary ecology, from community assembly [START_REF] Grime | Trait convergence and trait divergence in herbaceous plant communities: Mechanisms and consequences[END_REF] to biogeography [START_REF] Bergmann | Uber die verhaltnisse der warmeo-konomie der thiere zu ihrer grosse[END_REF] and evolution (Brown and Wilson 1956;Grant 1972). Geographical convergence -the phenotypic convergence of two species where their populations co-occur in the same geographic area-can reflect several evolutionary processes: hybridization [START_REF] Grant | Convergent evolution of Darwin's finches caused by introgressive hybridization and selection[END_REF][START_REF] Stern | The genetic causes of convergent evolution[END_REF], selection related to interspecific interactions (e.g. interspecific communication between territorial species: [START_REF] Cody | Convergent characteristics in sympatric species: a possible relation to interspecific competition and aggression[END_REF][START_REF] Secondi | Species-specific song convergence in a moving hybrid zone between two passerines[END_REF], adaptation to similar local environmental conditions [START_REF] Elmer | Adaptation in the age of ecological genomics: Insights from parallelism and convergence[END_REF][START_REF] Losos | Convergence, adaptation, and constraint[END_REF], or common plastic responses to these conditions (phenotypic plasticity: e.g. [START_REF] Dennis | Phenotypic convergence along a gradient of predation risk[END_REF]. Likewise, geographical divergence results from several mechanisms. Firstly, resource competition can promote divergence in ecological traits and generate ecological character displacement [START_REF] Schluter | Ecological Character Displacement in Adaptive Radiation[END_REF][START_REF] Pfennig | Character displacement: ecological and reproductive responses to a common evolutionary problem[END_REF]. Secondly, reproductive character displacement (RCD) can arise from selection to reduce interspecific reproductive interactions due to imperfect species recognition during mate choice [START_REF] Goldberg | Ecological and reproductive character displacement of an environmental gradient[END_REF]. Finally, agonistic character displacement (ACD) can evolve to reduce interspecific interference resulting from agonistic interactions between sympatric species [START_REF] Grether | The role of interspecific interference competition in character displacement and the evolution of competitor recognition[END_REF].

At the population level, phenotypic convergence and divergence depend on the interplay between various selective forces, and different body parts may show different responses depending on the sets of selective forces they are exposed to [START_REF] Shultz | The role of sexual and natural selection in shaping patterns of sexual dichromatism in the largest family of songbirds (Aves: Thraupidae)[END_REF]. Patterns of convergence and divergence may thus help deciphering the role of local adaptation, ecological competition and interspecific reproductive interactions in shaping the different dimensions of the phenotype (e.g. [START_REF] Langerhans | Shared and unique features of diversification in greater antillean Anolis ecomorphs[END_REF][START_REF] Jang | Convergent and divergent patterns of morphological differentiation provide more evidence for reproductive character displacement in a wood cricket Gryllus fultoni (Orthoptera: Gryllidae)[END_REF]. For example, widespread observations of ecological character displacement, the divergence in sympatry for traits involved in resource acquisition such as size or morphology of the feeding apparatus, provides powerful support for the role of competition in driving phenotypic evolution (reviewed in [START_REF] Grant | Ecological Character Displacement[END_REF][START_REF] Stuart | Ecological character displacement: glass half full or half empty?[END_REF].

Body coloration is a prime example of a trait subjected to various selective pressures, as it is involved in sexual signaling, avoiding (crypsis) or deterring (aposematism) predation, or regulating body temperature [START_REF] Hill | Bird coloration, Volume 2: function and evolution[END_REF][START_REF] Rudh | Adaptive colouration in amphibians[END_REF]. Colors of different body parts have different main functions. A range of situations exists from body parts usually concealed and exposed only during displays, such as the vocal sac in European tree frogs, whose coloration influence mate attraction [START_REF] Gomez | Support for a role of colour vision in mate choice in the nocturnal European treefrog (Hyla arborea)[END_REF], to body parts visible most of the time, assumed to be largely affected by environment-or predator-driven natural selection [START_REF] Stuart-Fox | Sexual selection, natural selection and the evolution of dimorphic coloration and ornamentation in agamid lizards[END_REF]. For this reason, investigating convergence and divergence patterns in a set of color traits including both sexual signals and nonsexual traits provides insight into the diversity of selective pressures that shape whole body coloration in animals. Previous studies have suggested that the functions of color patches in different body parts can predict their variation in sympatry: non-sexual colors may more often converge, while sexual signals may more often diverge in sympatry [START_REF] Stuart-Fox | Sexual selection, natural selection and the evolution of dimorphic coloration and ornamentation in agamid lizards[END_REF][START_REF] Gomez | Simultaneous crypsis and conspicuousness in color patterns: comparative analysis of a neotropical rainforest bird community[END_REF].

Patterns of trait convergence or divergence in the zone of sympatry between two related species are usually examined by comparing interspecific differences in phenotypic means in sympatric versus allopatric populations of both species [START_REF] Kawano | Character displacement in giant rhinoceros beetles[END_REF]. However, these differences may not reflect a specific evolutionary response to sympatry per se (i.e. to the presence of the other focal species) but simply emerge as a phenotypic response of each species to the environmental gradients experienced within its own range. Indeed, it is common for selective optima of phenotypic traits to vary across a species' range according to large-scale or small-scale environmental gradients [START_REF] Delhey | Reconciling ecogeographical rules: rainfall and temperature predict global colour variation in the largest bird radiation[END_REF][START_REF] Price | Background matching and disruptive coloration as habitat-specific strategies for camouflage[END_REF]. As a result, populations of each species may experience more similar environmental conditions in sympatry than in allopatry and thus be phenotypically more similar, independently of the presence of the other species. It is thus essential to account for environmental variations acting on the phenotype, at the scale of the species' distributions, to investigate divergence or convergence in nature [START_REF] Goldberg | Ecological and reproductive character displacement of an environmental gradient[END_REF].

Our aim here is to investigate patterns of divergence and convergence among sexual and non-sexual traits in a pair of related newt species. We used populations sampled in the wild to compare geographical variation in size, ornaments and coloration of Lissotriton helveticus and L. vulgaris within and outside their zone of sympatry. Specifically, we tested whether each trait diverged or converged in sympatry, and whether this pattern could be simply explained by phenotypic response to environmental gradients, or was also influenced by the presence of the other species. We also investigated whether traits involved in sexual signaling / communication were more likely to exhibit specific response to sympatry than traits assumed to be mainly under natural selection. Although by nature a correlative study, its sampling design, extensive geographical coverage and sophisticated analytical methods makes it a powerful approach to separate between alternative hypotheses of geographical variation in color phenotypes, even if the proximal mechanisms and selective agents remain to be tested experimentally.

MATERIALS AND METHODS

Biological models

The Smooth Newt Lissotriton vulgaris has a large distribution range across Europe (Figure 1, we follow [START_REF] Speybroeck | Species list of the European herpetofauna-2020 update by the Taxonomic Committee of the Societas Europaea Herpetologica[END_REF] for the taxonomy of the lineages that make up the Smooth Newt complex). From central France to northern Germany, one of its lineages shares a wide area of sympatry with the Palmate Newt L. helveticus, while the Palmate Newt has an allopatric range that reaches as far as southern France and north-western Iberia. In European newt species, interspecific reproductive interactions are common [START_REF] Babik | Nuclear markers, mitochondrial DNA and male secondary sexual traits variation in a newt hybrid zone (Triturus vulgaris x T. montandoni)[END_REF][START_REF] Pabijan | Isolation and gene flow in a speciation continuum in newts[END_REF] and in the sympatric zone, L. helveticus and L. vulgaris are frequently syntopic, and have similar reproductive phenology and ecological requirements during the breeding period [START_REF] Griffiths | Feeding niche overlap and food selection in smooth and palmate newts, Triturus vulgaris and T. helveticus, at a pond in Mid-Wales[END_REF][START_REF] Chadwick | Inter-and intraspecific differences in climatically mediated phenological change in coexisting Triturus species[END_REF].

We described the phenotypes of both species using several characters that we classified a priori as sexual or non-sexual, based on the biology of these organisms and the literature. The following characters were considered as sexual characters: (i) morphological ornaments (caudal filament, body and tail crests, hind-foot webbing, listed in Table 1) as they are only found in males, are displayed during courtship [START_REF] Halliday | The courtship of european newts: an evolutionary perspective[END_REF], and affect female choice in both species [START_REF] Gabor | Sequential mate choice by multiply mating smooth newts: females become more choosy[END_REF][START_REF] Cornuau | Information-content of morphological and behavioural sexual traits in the Palmate newt (Lissotriton helveticus)[END_REF]. (ii) The conspicuous yellow-orange coloration with black spots, found on the belly of both sexes, because it has been reported to be involved in female choice [START_REF] Secondi | Male attractiveness is influenced by uv wavelengths in a newt species but not in its close relative[END_REF]Secondi and Théry 2014; but see [START_REF] Cornuau | Multiple signals in the palmate newt: ornaments help when courting[END_REF]) and to influence male mate choice in a related species [START_REF] Lüdtke | Choosy males court both large, colourful females and less colourful but responsive females for longer[END_REF]. Ventral coloration is probably not an aposematic signal in our species (contra other Salamandridae, see [START_REF] Brodie | Salamander Antipredator Postures[END_REF] since there is no published evidence of toxicity and adults are consumed by a large number of predators [START_REF] Rosa | Field observation of foraging behavior by a group of adult diving beetles Agabus (Gaurodytes) bipustulatus preying on an adult Lissotriton boscai[END_REF][START_REF] Parry | Predation of newts (Salamandridae, Pleurodelinae) by Eurasian otters Lutra lutra (Linnaeus)[END_REF][START_REF] Miró | Rapid amphibian community recovery following removal of non-native fish from high mountain lakes[END_REF]. (iii) Finally, we also classified caudal coloration as a trait under sexual selection because, although its role in mate choice has not been studied specifically, it is sexually dimorphic and the tail plays an overall important part during courtship [START_REF] Halliday | The courtship of european newts: an evolutionary perspective[END_REF]. On the contrary, dorsal coloration was assumed to be under natural, but not sexual, selection, because both sexes of both species share a dull brown coloration broadly matching the pond substratum, making the newt difficult to detect. It should also be noted that ornaments (crests, webbing and tail filament) are only expressed during the breeding season while dorsal and ventral coloration seem more stable over the year. As each of these traits is likely to be the target of some form of selection, we did not expect any of them to have a purely neutral evolution, though indirect selection via genetic correlations among traits may happen.

Sampling

We sampled L. helveticus and L. vulgaris (belonging to the northern lineage of L. v. vulgaris, see [START_REF] Pabijan | Isolation and gene flow in a speciation continuum in newts[END_REF] in the allopatric and sympatric part of their distribution ranges (Figure 1). Adult newts were caught by dipnetting in ponds during the breeding period from March 2012 to May 2014. All newts were anaesthetized prior to manipulation by immersion in Tricaine methane sulphonate (MS222, 0.2 g/l) for 5 minutes. After the measurements, newts were released at their site of capture. All individuals that we monitored fully recovered from anesthesia. We sampled 55 sites in total, 32 in sympatry, 15 in the allopatric distribution of L. helveticus and 8 in the allopatric distribution of L. vulgaris (location and number of individuals sampled in both sexes for each sampling site in Table S1). In the allopatric areas we caught 59 male and 70 female L. vulgaris, and 117 male and 100 female L. helveticus. In the sympatric zone, we captured 137 male and 127 female L. vulgaris, and 311 male and 237 female L. helveticus. We excluded males without fully developed sexual ornaments (crests, palmation). Final sample size was 54 male and 70 female L. vulgaris and 101 male and 100 female L. helveticus in allopatry and 124 male and 127 female L. vulgaris and 309 male and 237 female L. helveticus in sympatry.

Morphology, pattern and spectrometry measurements

Using a digital caliper, we measured snout-to-vent length and tail height in both sexes, plus the caudal filament length in males. Each measurement was taken twice and the mean was used in further analyses. For males, we also described the sexual ornaments by coding the extent of the hind-foot web, the shape of the dorsal and caudal crest, the number of dark spots on the belly, and the number and size of the dark spots on the throat and the flank (scoring of the traits detailed in Table 1, pictures of males of both species in Figure S1).

The morphological variables were all correlated to snout-to-vent length, as it is a measurement of body size in amphibians. We used two methods to remove body-size effects on the other morphological traits. For females, we only had one morphological trait (tail height) in addition to snout-to-vent length, and we used the residuals of the linear regression of tail height on snout-to-vent length to make tail height independent of body size. For males, we had a set of 11 traits, so we used a Principal Component Analysis (PCA) to reduce the number of variables to a few independent synthetic traits, while accounting for size effects in the first principal component (see below).

For coloration, we measured the reflectance spectra between 300 and 700nm of dorsal, ventral and caudal body parts in all individuals. Measurements were taken on wet animals, and two measurements were taken for each body part. We averaged and smoothed the reflectance spectra for each individual with the R package pavo [START_REF] Maia | pavo : an R package for the analysis, visualization and organization of spectral data[END_REF]. Flank coloration was not measured, as their upper and lower coloration appear to be the same as dorsal and outer (lateral) ventral coloration respectively, at least to the human eye. However, we quantified the number and the size of dark spots on the flank (see above). For ventral coloration, we measured the vividly-colored central band of the belly, which is involved in reproductive interactions in newts (Secondi & Théry, 2014;[START_REF] Lüdtke | Choosy males court both large, colourful females and less colourful but responsive females for longer[END_REF]. The exact location of the measurements on the body is described in Figure S1 (for the ventral coloration of L. vulgaris we made sure to measure the vivid coloration between the black spots).

We used a Jaz spectrometer (Ocean Optics), a deuterium-halogen light source (Ocean Optics DH-2000), and a coaxial optic fiber (Avantes FCR-7UV200-2-45-ME). The probe was held with an angle of 45°. Reflectance spectra of each body part (Figure 2) are multidimensional traits, and from each of them we were able to extract several variables: the hue in the visible range (401-700nm), the saturation and brightness in the UV range (300-400 nm) and in the visible range separately. We did not describe the full 300-700nm range with only one variable because the spectra were bimodal (Figure 2), and because there is evidence that females use UV to assess males (Secondi & Théry, 2014). We did not estimate hue for dorsal coloration because we observed no peaks in the spectra, and overall a very low reflectance (<6%, see Figure 2). The most appropriate color variables for each part of the spectrum were selected among those described by [START_REF] Montgomerie | Analyzing colors[END_REF], and were given the same name (algorithm for each variable described in Supplementary methods). We measured brightness with B2, in UV (B2_UV) and visible range (B2_visible); saturation in the UV range using S1 (S1_UV) and in the visible range using S6 (S6_visible). For the ventral coloration, we chose another saturation index (S9), more appropriate to describe carotenoid-based coloration, which probably applies to newt ventral coloration [START_REF] Kopecký | Sex differences in ventral side colouration of Alpine newts[END_REF]. We measured hue in the visible range with the H3 variable (H3_visible). In summary, we extracted five (ventral and caudal colorations) or four variables (dorsal coloration) describing the reflectance spectrum in the visible and UV ranges. We do not know of any study assessing whether capture and handling affects coloration in newts but we did not notice any change in coloration during the process. In addition, the photos of animals taken just after capture and after handling and measurements did not show obvious differences. We are thus confident that we recorded live coloration accurately and further note that all populations were treated in the same way, ensuring than none of the reported effects result are artefacts due to capture and handling.

We log-transformed several coloration traits before analysis to ensure normality (specified in Table S2). We also standardized all the traits before analysis (mean-centered and divided by the standard deviation) to facilitate comparison between model estimates. Finally, we measured the correlation between coloration traits in both sexes, as coloration traits are known to often coevolve (Figure S2). The correlations between traits describing different body parts were generally low, but they were higher between traits describing the same body parts.

Environmental factors

We considered both large-scale and local environmental factors that can affect the expression of traits in newts. We extracted 19 bioclimatic variables from WorldClim v2 [START_REF] Fick | WorldClim 2: new 1-km spatial resolution climate surfaces for global land areas[END_REF], with 1km resolution, and used the first two axes of a PCA to summarize climatic variation among sites. The first axis accounted for 44% of the variance, and separated warm sites with high winter rainfall (negative side) from cold sites with high summer rainfall (positive side; Table S3). The second axis accounted for 24% of the variance, and described a gradient of temperature seasonality (high seasonality for negative values of PC2). We also included altitude (1-km resolution, Danielson and Gesch 2011) in our models. At a local scale, we quantified variation of water transparency by measuring the water transmission spectrum for each pond, and averaging transmission over the whole spectrum (method described in [START_REF] Secondi | Variability of surface and underwater nocturnal spectral irradiance with the presence of clouds in urban and peri-urban wetlands[END_REF]. Mean values of environmental variables in the sympatric and allopatric zones are presented in Table S4.

Analysis 1: investigating patterns of convergence/divergence

The aim of this first analysis was simply to detect patterns of divergence and convergence in sympatry, without trying to decipher the influence of environmental gradients and sympatry itself. We used Linear Mixed-effect Models, implemented in 'lme4' R package [START_REF] Bates | Fitting Linear Mixed-Effects Models using lme4[END_REF].

The model (1) included species identity, context (sympatry versus allopatry) and their interaction as fixed factors, and sampling site as a random factor. Species identity tested for difference in trait value between species, while context tested for difference in trait value between allopatric and sympatric zone, and their interaction tested if the two species showed a similar response to sympatry or not. A significant interaction term indicates either convergence or divergence (the two species react differently to sympatry hence are more similar, or less similar, in sympatry than in allopatry).

(1) Trait ~ species * context + sampling site We ran one model for each trait and sex. Sexes were analyzed separately because we did not expect a similar response to context in both sexes and species, and a three-way interaction would have been uninterpretable. We did not include the year of sampling in the models, as this variable was confounded with geographic pattern (most regions were only sampled once, see details in Figure 1). However, we did not expect sampling date to affect the result of the model, because between-year phenotypic variations should be limited compared to phenotypic differences observed at continental scale. We investigated homoscedasticity and normality of the traits using diagnostic plots of models' residuals, but we did not find patterns indicating violation of the linear model hypotheses.

Convergence and divergence in sympatry can be inferred from the species * context interaction in linear models, but this requires a new calculus for each model. To provide a global vision of the convergence/divergence patterns observed on all the characters measured, we computed for each trait and sex an index describing the pattern observed in sympatry. We the predicted mean value of a trait, after removing the effect of other variables included in the model. For Analysis 1, the marginal means were equal to the mean values of species traits because we did not include additional variables in the model. If the difference is smaller in sympatry, the index is negative and the species have converged. If the sympatric difference is larger, the index is positive and the species have diverged. This index was not subjected to any statistical modelling (since the significance of the sympatry /allopatry effect was tested by means of the context and species * context effects) but was computed for illustrative purpose only.

In order to limit Type-I errors due to repeated testing of the same hypothesis on independent datasets, we used a cross validation procedure: we randomly divided the full dataset into two non-overlapping sub-datasets (half of the data in each), and we repeated the analysis on each. We only retained the effects that were significant in the global model and in both sub-datasets.

Analysis 2: Testing the effect of sympatry and environmental factors on trait variation

In this second analysis, we assessed the contribution of the environment on phenotypic variation, and tested whether responses to environmental variations entirely explained the divergence and convergence patterns in sympatry detected in Analysis 1. For each sex and trait separately, we first created a full model, which included the fixed and random factors already present in model ( 1), plus all environmental variables, and their interaction with species identity. This model (2) fitted species-specific responses to each environmental variable for each trait.

The full model (2) thus reads:

(1) Trait ~ species * (context + Bioclimatic axis 1 + Bioclimatic axis 2 + altitude + water transmission) + sampling site We applied a model selection procedure in order to reduce the number of predictors/explanatory variables. Using the dredge function of MuMIn package [START_REF] Barton | Package "MuMIn[END_REF] we performed the models corresponding to all possible combination of the fixed environmental effects. We forced the model selection procedure to keep species identity, context, and their interaction in the model. Then we selected the simplest model with a deltaAICc of less than 2 compared to the model with the lowest AICc. Finally, we computed the index of convergence/divergence in a similar way as in the first analysis for each trait and sex, but this time the marginal means were calculated after removal of the environmental effects. Thus, the part of the difference in a trait observed in sympatry that was due to environmental variables, as well as their possible interactions with species identity, was removed, leaving only the difference brought about by the species-by-context interaction term. The corrected index is positive (respectively negative) when the accentuation of species differences in sympatry (vs allopatry) is larger (respectively weaker) than predicted by large-scale environmental gradients.

We performed the same cross-validation procedure as in Analysis 1. Likewise, we checked the validity of each model using the diagnostic plots of the residuals, which did not show any patterns indicating violation of the linear model hypotheses. We also measured the Variance Inflation Factor (VIF) for each model, to estimate the impact of collinearity between variables [START_REF] Mansfield | Detecting Multicollinearity[END_REF]. Finally, the pattern of convergence/divergence observed in sympatry may be partially explained by the non-independence of the phenotypic measurements between populations due to gene flow, thus generating spatial autocorrelation among populations. To test this hypothesis, we performed Moran's tests, using the spdep R package [START_REF] Bivand | Thermoregulation in reptiles, a factor in evolution[END_REF], to evaluate the spatial autocorrelation of the residuals.

RESULTS

PCA on male morphological traits

The first three PCs on male ornamental traits accounted for respectively 67%, 9% and 8% of the variance and were retained for further analyses. The first axis separated the two species based on the size of the caudal filament, the shape of the foot webs and the crest, the number and size of spots on the throat, the number of spots on the belly and the size of spots on the flank (Table 1, see Figure S3 for a plot of individuals' scores on the first axes). The second axis separated individuals with few vs many dark spots on the flanks and the third axis separated large individuals with many dark spots on the flanks from small individuals with few spots.

Patterns of divergence and convergence in sympatry

The results of model ( 1) indicate that the two Lissotriton species differ by many traits (9 male and 6 female traits), most of which presented significant species-by-context interactions (7 for males and 8 for females, model results presented Figure 3A and Table S2). Our index of divergence/convergence indicated the existence of several patterns (Figure 3A). Male ornamental traits (summarized as PC1-3) and female morphological traits (SVL, tail height) did not show any signs of convergence or divergence. Dorsal and caudal coloration traits tended to converge in sympatry, while a pattern of divergence was only observed for the variables describing the coloration of the ventral band (except brightness in the visible range). Overall, males and females showed similar patterns of convergence and divergence.

Responses to environmental variables and their impact on divergence and convergence

Environmental effects may result in convergence (i) when both species react similarly to an environmental variable that takes different values in sympatry and in allopatry or (ii) when species react differently to the same variable in such a way that their difference is decreased in sympatry. For these reasons, the patterns visible in Figure 3A may not reflect an actual response of species' phenotypes to sympatry, but rather to the environment. Indeed, model (2) revealed many significant effects of environmental variables on male and female traits (results of model (2) in Table S5). After cross-validation, we found that coloration and morphology co-varied mainly with large scale gradients, bioclimatic variables and altitude (respectively 8 and 7 traits correlated in males and females, Table S5), while local environmental conditions, water transmission, had a generally smaller influence (only one trait in males). Focusing on the effects that were retained for both species, we found that for variation in male morphology, PC1 (which differentiates the two species) was mainly influenced by the second bioclimatic axis (Table S5) and water transmission, while PC2 (which differentiates individuals with low and high number of spots on the flanks) was influenced the two bioclimatic axes. For coloration, we found that dorsal brightness in the UV-spectrum in both sexes and dorsal saturation in the UV spectrum in females both decreased in regions with less thermal contrasts between seasons (axis 2). Dorsal brightness in the visible and UV spectrum and dorsal saturation also increased with altitude in females. However, since dorsal brightness and dorsal saturation are strongly correlated (Table S2), the effect of altitude found in females could be driven by one of these two traits only. For the ventral parts, saturation in the visible spectrum increased in males and females as climate became less seasonal (axis 2).

The two species also showed differential responses to some environmental variables (interaction species:environmental variables Table S5). In particular, L. vulgaris had stronger responses to altitude than L. helveticus. Caudal saturation (S6_visible) and caudal brightness (B2_visible) in the visible spectrum decreased with altitude in both sexes for L. vulgaris, while these traits were not affected by altitude in L. helveticus. Similarly, male ventral saturation (S9) decreased with altitude in L. vulgaris but not L. helveticus. However, the pattern was opposite for male ventral hue (H3_visible): it increased with altitude in L. helveticus and not L. vulgaris.

Patterns of convergence and divergence obtained in the first analysis were re-examined based on the model including environmental variables (see Table S5 for model (2) results and Figure 3B for the pattern of convergence/divergence after controlling the effects of environmental variables). To facilitate the interpretation of Figure 3, we will detail one example in which the inclusion of environmental responses makes a visible difference, the S6_visible variable describing caudal coloration in females, in which a strong convergence signal is uncovered only when environmental variables are taken into account. For this variable the average trait difference (L. vulgaris -L. helveticus) in sympatry is small (0.5 on standardized scale) and comparable in absolute value to the difference in allopatry (-0.74); this is why our divergence/convergence index is close to zero, without taking environment into account (0.5-0.74=-0.24 in Figure 3A, Table S2). However, in the model with environmental predictors (Table S5), this variable is significantly sensitive to altitude and to climatic gradients (PC2), and these responses are species-specific (significant interactions). Based on these responses, and on the mean environmental values of the sympatric and allopatric zones (Table S4), environmental gradients would predict a phenotypic difference between the two species of 2.91 in allopatry, and of 2.91 in sympatry, the latter much larger than the observed value. The model fits the data by adding a very large species-by-context interaction (-3.51) that reduces this difference specifically in the sympatric zone, resulting in a very negative divergence index (0.6-2.91=-2.31 in Figure 3B). This means that the phenotypic differences between the two species in sympatry is much lower than what would be expected based on their responses to environmental gradients.

Looking at all the variables, however, the inclusion of environmental factors did not overall strongly modify the observations on convergence and divergence obtained in the first analysis (Figure 3B). The effect of environmental factors on coloration, although significant, explained a relatively low percentage of the whole variance (examples of relationship between coloration and environment in Figure S4). For three out of the twelve traits that significantly converged in the first analysis, the species-context interaction was no longer significant, though still indicating convergence (in females : caudal B2_visible and S1_UV, in males ventral H3_visible, Figure 3B), suggesting that the initial pattern was largely explained by responses to the environment. For one other trait (caudal S6_visible in females, example detailed above), the pattern of convergence was on the contrary reinforced when the environmental effects were taken into account (Table S5).

Patterns of divergence suggested by the first analysis were maintained for two ventral colorimetric variables in the second analysis, S9_visible in males and S1_UV in females. The divergence of the last trait in males (ventral H3_visible), was qualitatively maintained but not statistically supported when accounting for environmental variables as the species-context interaction became non-significant. In Figure 4 we illustrated the variation in the two traits found to be divergent in all models: we displayed the raw data in sympatry and allopatry along with predictions for models including environmental variations only. The comparison between these two graphs indicates if divergence was driven by one or both species. For male ventral coloration (S9), the interspecific difference in raw values increased from 0.23 in allopatry to 1.41 in sympatry (Table S2). This enhanced divergence between raw values and values predicted by environmental variation came from the reduced saturation of L. helveticus in sympatry. For female ventral coloration (S1_UV), the interspecific difference in raw values increased from 0.09 in allopatry to 0.85 in sympatry (Table S2). This time the divergence arose from the higher saturation of L. helveticus in sympatry. Thus, for both traits, divergence was the result of a change in L. helveticus, while L. vulgaris traits exhibited only small variation between sympatry and allopatry (Figure 4). Moran's tests for spatial correlation of the residuals were not significant for any model (p>0.05, Table S6). The VIF was low for most simple effects but high in case of interactions (Table S7). Because collinearity inflates the standard errors, but does not bias the coefficients, the corresponding tests are expected to be less often significant [START_REF] Morrissey | Multiple regression is not multiple regressions: the meaning of multiple regression and the non-problem of collinearity[END_REF]. A high VIF may also indicate overdispersion, but diagnostic plots of residuals did not show any evidence of it. The results presented above are thus robust, although we may have missed some significant interactions due to a lack of power.

DISCUSSION

Our analyses revealed that sympatry affected the phenotype of L. vulgaris and L. helveticus differently depending on the body part investigated: most of the dorsal and caudal coloration traits converged in sympatry in both sexes, while divergence was observed only on two variables describing ventral coloration, one in males one in females, and no difference between sympatry and allopatry was detected for morphological traits. Divergence was thus restricted to a sexual character (ventral coloration), whereas convergence was observed on both sexual and nonsexual characters (respectively caudal and dorsal colorations). When accounting for environmental variations, the patterns of convergence/divergence were maintained, though environmental variables had significant effects on many traits. Finally, the few ventral colorimetric variables showing divergence in sympatry were not explained by environmental gradients and thus constitute likely cases of character displacement.

Environmental effects on newt phenotype mostly lead to convergence

Our analyses revealed that the phenotype of the two Lissotriton species was influenced by largescale gradients more than local water color. This result contrasts with growing evidence for strong effects of local conditions on phenotype in newts [START_REF] Secondi | Water turbidity affects the development of sexual morphology in the palmate newt[END_REF] and other taxa [START_REF] De Abreu | Spatial and environmental correlates of intraspecific morphological variation in three species of passerine birds from the Purus-Madeira interfluvium, Central Amazonia[END_REF][START_REF] Price | Background matching and disruptive coloration as habitat-specific strategies for camouflage[END_REF]. It is possible that water color fluctuates too much in time (e.g. reduced transparency after rainfall) or space (i.e. large differences between neighboring sites) to trigger stable plastic and/or evolutionary responses [START_REF] Sasaki | The evolutionarily stable phenotype distribution in a random environment[END_REF].

Environmental selection is known to influence the evolution of both sexual and nonsexual traits [START_REF] Andersson | Sexual selection, natural selection and quality advertisement[END_REF][START_REF] Endler | Natural and sexual selection on color patterns in poeciliid fishes[END_REF], leading to phenotypic convergence between sister species [START_REF] Wilkens | Convergent adaptations to cave life in the Rhamdia Laticauda catfish group (Pimelodidae, Teleostei)[END_REF] or distantly related taxa [START_REF] Mahler | Exceptional convergence on the macroevolutionary landscape in island lizard radiations[END_REF] exposed to the same conditions. In many vertebrates, dorsal coloration has a critical role for thermal physiology [START_REF] Clusella-Trullas | Thermal melanism in ectotherms[END_REF][START_REF] Alho | Increasing melanism along a latitudinal gradient in a widespread amphibian: local adaptation, ontogenic or environmental plasticity?[END_REF] and crypsis [START_REF] Rosenblum | Convergent evolution and divergent selection: lizards at the White Sands ecotone[END_REF][START_REF] Toledo | Colors and some morphological traits as defensive mechanisms in anurans[END_REF][START_REF] Rudh | Adaptive colouration in amphibians[END_REF], which could explain the influence of climate and altitude on newts' dorsal coloration. However, we did not observe a decrease in dorsal brightness in cooler environments (Bogert's rule; Bogert 1949), which would be expected under the thermal adaptation hypothesis. Crypsis is another plausible function of dorsal coloration. Unfortunately, quantifying large-scale variation in predation pressure is a daunting task at such a continental scale, so we were unable to directly evaluate the potential for adaptive geographic variation in coloration in relation to predator avoidance. For the sexual traits, climatic variables could also correlate with conditions of transmission of sexual signals in the breeding habitat, and variable access to trophic resources leading to differential allocation of resources to sexual traits [START_REF] Snell-Rood | The effect of climate on acoustic signals: Does atmospheric sound absorption matter for bird song and bat echolocation?[END_REF][START_REF] Macías-Ordóñez | Macroecology of Sexual Selection: Large-Scale Influence of Climate on Sexually Selected Traits[END_REF]. We wish to stress that climate per se is not necessarily the causal agent driving variation in coloration but climatic gradients might also capture large-scale variation in vegetation, predator and prey communities, parasites, pathogens and other biotic or abiotic factors that can affect the selective optima of color traits.

Despite their strong effects on body coloration, environmental variables did not explain most of the convergence of dorsal and caudal colors. It is unlikely that the observed convergence arose from introgression because there is no detectable interspecific gene flow between these two species [START_REF] Arntzen | Rare hybridization and introgression in smooth and palmate newts (Salamandridae: Triturus vulgaris and T. helveticus)[END_REF]. Convergence due to agonistic interaction is also unlikely. This type of convergence is known to arise in territorial species, because shared signals may help individuals of both species to defend their territories against both conspecifics and heterospecific competitors [START_REF] Cody | Convergent characteristics in sympatric species: a possible relation to interspecific competition and aggression[END_REF][START_REF] Grether | The role of interspecific interference competition in character displacement and the evolution of competitor recognition[END_REF]. However, although reproductive interferences during courtship may occur among male newts [START_REF] Halliday | Intrasexual selection and alternative mating behaviour[END_REF][START_REF] Hardy | Animal Contests[END_REF], Lissotriton newts are not territorial [START_REF] Giacoma | Phylogeny of the salamandrid genus triturus[END_REF]. A more likely explanation for residual convergence in dorsal and caudal colours is an environmental selection, not captured by our -mostly climatic-environmental models, that differs in the sympatric zone, and to which species respond by adopting similar dorsal and caudal colorations. It may be predators (as mentioned above), that have been shown to explain patterns of phenotypic convergence in other species [START_REF] Dennis | Phenotypic convergence along a gradient of predation risk[END_REF], but also vegetation structure and composition, prey types, among others.

In conclusion, while we cannot definitively identify all environmental drivers of phenotypic variation in Lissotriton, our results support that similar responses of the two species to shared environmental factors is the most likely cause of convergence for some traits in sympatry. Common garden experiments would be required to establish if these common responses to environment reveal convergent local adaptation or shared plastic responses.

Evidence for character displacement in males and females sexually-selected ventral colors

We found a pattern of divergence in two traits related to ventral coloration in males and females (S9_visible and S1_UV respectively). This pattern was maintained after accounting for environmental effects, and thus constitute a probable case of character displacement [START_REF] Goldberg | Ecological and reproductive character displacement of an environmental gradient[END_REF][START_REF] Adams | Analysis of character divergence along environmental gradients and other covariates[END_REF][START_REF] Grether | The role of interspecific interference competition in character displacement and the evolution of competitor recognition[END_REF]. Divergence, like convergence, could in theory also be explained by plastic responses to unaccounted environmental factors if the two species present species-specific -and opposite-plastic responses to these factors. However, this explanation is unlikely in our situation. Firstly, contrary to the case of convergent responses, it is difficult to imagine how an unmeasured environmental factor could diverge in sympatry when the two species often occur in the same ponds. If the divergence was due to plasticity, it should involve plastic responses to the same factor in opposite directions in two closely related species, a situation for which we know of no empirical example. Secondly, the fact that the traits exhibiting divergence are known sexual signals and diverge in sympatry suggests that this divergence is a direct response to the presence of the other species and that it reduces interspecific interactions. Such divergence in sexual signals in response to the presence of a closely related species typically involve a genetic basis [START_REF] Servedio | The Role of Reinforcement in Speciation: Theory and Data[END_REF][START_REF] Ortíz-Barrientos | Evidence for a One-Allele Assortative Mating Locus[END_REF][START_REF] Mcgraw | Highdimensional variance partitioning reveals the modular genetic basis of adaptive divergence in gene expression during reproductive character displacement[END_REF], and to our knowledge there are no reports of divergence in sexual characters in related species explained by species-specific plastic responses. We thus believe that the residual unexplained divergence in our models is not the results of unaccounted environmental factors but can rather be explained by sympatry per se and is unlikely to be plastic, even if common garden experiments would be required to formally exclude this possibility.

Because ventral colors are mostly visible during courtship, character displacement is probably related to communication, either for mate attraction between sexes (it would then be a reproductive character displacement, RCD), or within sexes, as part of signaling towards competitors (agonistic character displacement, ACD).

The first possible advantage of divergence in sexual signals in sympatry is to reduce the production of unfit hybrids. Hybrids obtained experimentally between L. vulgaris and L. heleveticus are viable, despite being sterile, yet F1-hybrids are extremely rare in nature [START_REF] Griffiths | A natural hybrid newt, Triturus helveticus × T. vulgaris , from a pond in mid-Wales[END_REF][START_REF] Arntzen | Rare hybridization and introgression in smooth and palmate newts (Salamandridae: Triturus vulgaris and T. helveticus)[END_REF][START_REF] Schlüpmann | Nachweis eines Freilandbastardes von Teichmolch (Triturus vulgaris) und Fadenmolch (Triturus helveticus)[END_REF]. This indicates that interspecific fertilization rarely occurs in natural conditions and that hybridization avoidance is already strong between the two species, both suggesting that hybridization avoidance has only a small potential impact on divergence. Yet, species recognition is not perfect in males and females of the two species : males of both species frequently court heterospecific females [START_REF] Roberts | Reproductive isolation in Smooth and Palmate newts (Triturus vulgaris vulgaris and T. helveticus helveticus)[END_REF], and experimental tests showed a low interspecific recognition for female L. helveticus and male L. vulgaris in captivity [START_REF] Secondi | Olfactory and visual species recognition in newts and their role in hybridization[END_REF](Secondi et al., , 2014)). We thus suggest that RCD evolved under selection for enhanced interspecific recognition, and associated gains in time and energy (otherwise wasted in interactions that eventually do not result in effective mating), in sympatry. This situation may also explain why divergence affects both sexes in newts, while it is commonly observed in males only (see [START_REF] Dyer | Reproductive character displacement of epicuticular compounds and their contribution to mate choice in Drosophila subquinaria and Drosophila recens[END_REF]. In males, mating attempts are costly because of energy and time invested in complex displays, but also because spermatophores used for transferring sperm to the female are a highly limited resource that should not be wasted [START_REF] Verrell | Limited male mating capacity in the smooth newt, Triturus vulgaris vulgaris (Amphibia)[END_REF]. During heterospecific courtship, males will pay this cost without the benefits of mating.

However, the most divergent males could pay a lower cost, because females may identify them more easily and interrupt courtship earlier. Such a situation would select for a divergence of male coloration in sympatry. Divergence in females could be due to genetic cross-sex correlations, but this explanation is not convincing in our case, as the diverging traits were different between sexes. Rather this pattern could stem from the mutual mate choice observed in newts [START_REF] Roberts | Reproductive isolation in Smooth and Palmate newts (Triturus vulgaris vulgaris and T. helveticus helveticus)[END_REF][START_REF] Lüdtke | Choosy males court both large, colourful females and less colourful but responsive females for longer[END_REF]. This could happen in two non-exclusive situations: (i) if males preferentially court females with more recognizable coloration, while females with intermediate coloration have fewer opportunities and may fail to obtain spermatophores, or (ii) if males do not discriminate well between species and females pay a cost when repeatedly courted by heterospecific males. This cost would select for more recognizable coloration in females, reducing the error rate of males (i.e. initiating heterospecific courtship les often) and the associated cost for the females. These conditions are likely to be fulfilled given what we know of the breeding and courtship habits of these newts. Hence, a mutual benefit of enhanced interspecific recognition in both sexes is a likely explanation for divergence of ventral coloration in sympatry. Direct empirical tests are needed to validate this hypothesis by directly measuring sexual preferences.

Another explanation for the divergence of coloration in sympatry could be male competition through interspecific reproductive interference. In Lissotriton, males sometimes interfere in the courtship of conspecific males [START_REF] Halliday | Intrasexual selection and alternative mating behaviour[END_REF][START_REF] Hardy | Animal Contests[END_REF], and, in males, the pattern of divergence could thus be a case of agonistic character displacement (ACD) -i. e. to avoid unnecessary interference with heterospecific males. It should be noted that, so far, we have no empirical evidence of interference during courtship between males of different newt species in nature, but nothing suggests it cannot happen. On the other hand, ACD is unlikely to explain the pattern observed in females, as reproductive interference has not been reported in this sex.

The divergence observed in ventral colors in sympatry, whether in males or in females, was driven by changes in L. helveticus only. Asymmetry can be caused by either unidirectional hybridization or asymmetrical cost of interspecific reproductive or agonistic interactions [START_REF] Smadja | Asymmetrical reproductive character displacement in the house mouse[END_REF][START_REF] Lemmon | Diversification of conspecific signals in sympatry: geographic overlap drives multidimensional reproductive character displacement in frogs[END_REF]. None of these scenarios can be excluded for now. One potential cause of asymmetrical cost is asymmetry in numbers: the more abundant taxon should pay the lower cost, as encounters with the other taxon are rarer. However, in our case, this is unlikely, as we sampled a higher number of L. helveticus in most sympatric sites (24 out of 33). Further investigations are required to investigate the origin of the asymmetrical responses of the two Lissotriton studied.

If character displacement affects coloration in these species, one might wonder why we did not detected divergence in all coloration traits involved in signaling. In fact, while RCD and ACD can affect any trait as soon as it increases interspecific recognition, not every trait potentially involved in signaling needs to exhibit divergence in a given species pair. First, although traits involved in interspecific recognition are usually sexual [START_REF] Ryan | Species recognition and sexual selection as a unitary problem in animal communication[END_REF], not all sexual traits signal species identity. For example some of them may be constrained by a shared female preference, maintained in both species as a way to assess male physical condition (Pérez i De [START_REF] Pérez I De Lanuza | Colours of quality: Structural (but not pigment) coloration informs about male quality in a polychromatic lizard[END_REF]. This might explain why some sexual traits other than ventral coloration did not diverge in our system: they may simply not be used for interspecific recognition. Another possibility is that these sexual traits indeed allow species recognition, but are already strongly divergent in allopatry, thus limiting the potential benefits of further divergence. This hypothesis is particularly likely for the caudal filament and extent of webbing, sexual ornaments that are only found in L. helveticus, see Figure S1). A similar argument can also apply, to some extent, to different variables describing the same color. We observed that among colorimetric variables describing ventral coloration, some did not diverge or even converged in sympatry. This may be because the change in only one component (e.g. the hue) suffices to alter dramatically the perceived ventral coloration, making changes in other components redundant. Amphibians are known to discriminate subtle variations of coloration, even in dim light conditions (e.g. at night, [START_REF] Gomez | Support for a role of colour vision in mate choice in the nocturnal European treefrog (Hyla arborea)[END_REF], and the enhanced difference in saturation and hue in sympatric populations can thus certainly be perceived by newts.

Conclusion

Our study illustrates how the colors of different parts of the body, subject to different combinations of selection pressures, can follow a diversity of evolutionary trajectories, some convergent, others divergent, when two species meet each other in a sympatric zone. An important point is that, because by definition sympatry means that the two species are confronted to similar environments, at least with respect to large-scale environmental gradients (e.g. driven by latitude), convergence can be considered the "null" expectation for colors predominantly involved in response to environment-related selection -and we indeed frequently observed convergence in dorsal and caudal colors in our newt species. In such cases, the inclusion of environmental variables that potentially differ among regions in the model, as we did, may help to isolate some of the potential drivers of convergence -though it will be difficult to capture them entirely. On the other hand, the opposite pattern, divergence in sympatry, if it resists the inclusion of environmental variables in the model, suggests that the interaction between the two species has specifically resulted in selection for dissimilarity in the corresponding traits.

Our observations on newts are consistent with earlier studies suggesting that, in sympatry, non-sexual colors more often converge and sexual signals more often diverge [START_REF] Stuart-Fox | Sexual selection, natural selection and the evolution of dimorphic coloration and ornamentation in agamid lizards[END_REF][START_REF] Gomez | Simultaneous crypsis and conspicuousness in color patterns: comparative analysis of a neotropical rainforest bird community[END_REF]. For the most constantly exposed color patches, potentially involved in thermal regulation or crypsis, we observed convergence (sometimes explained by large-scale environmental gradients) while divergence was only observed in traits of usually concealed body parts (i.e. ventral colors) involved in mate choice. It is important to note however, that RCD or ACD (the most likely sources of selection for dissimilarity in sexual traits), are not expected to apply equally to all sexual traits, because of potential redundancy among them as means of species recognition, and other sources of (non-sexual) selection that may simultaneously affect them. Newt tails, for example, are both sexually selected and visible Table 1. Description of the morphological and colorations included in the study. Is specified if these traits are expected to be sexual or not (supporting literature in the Material and Methods section of the manuscript). * indicates morphological and patterns traits only measured on males and included in the Principal Component Analysis (PCA). Is also specified the correlations between these traits and the first three PCA axes (values in bold indicate correlations above 0.5). Overall the first axes accounted for 67% of the total variance, the second axis for 9% and the third axis for 8%.

  calculated this index following the formula: Index = |Marginal mean Lh sympatry -Marginal mean Lv sympatry | -|Marginal mean Lh allopatry -Marginal mean Lv allopatry | With Lh = Lissotriton helveticus and Lv= Lissotriton vulgaris. The marginal mean corresponds to

Figure 1 .

 1 Figure 1. Map of the distribution ranges of Lissotriton helveticus (Lh) and L. vulgaris (Lv) with the location and the date of sampling of the sites included in the study. Sampling size in each sympatric and allopatric zones is indicated between brackets. Species' distributions were obtained from IUCNRed List maps (IUCN 2018).

Figure 2 .

 2 Figure 2. Mean spectral reflectance of female (a) and male (b) dorsal, ventral and caudal patches for Lissotriton vulgaris and Lissotriton helveticus originating from the sympatric and the allopatric zone. Dashed lines and shaded area represent the standard error around each spectrum. The black vertical dashed lines separate the UV range (left) from the visible range (right).

Figure 3 .

 3 Figure 3. Score of the convergence / divergence index for morphological and color variables in males and females of Lissotriton helveticus and L. vulgaris obtained from (A) analysis 1, without inclusion of environmental variables, or (B) analysis 2, with environmental variables included. Index = |marginal mean LH sympatry -marginal mean LV sympatry|-|marginal mean LH allopatry -marginal mean LV allopatry| (LH = L. helveticus, LV= L. vulgaris).For the analysis without environmental variables, the marginal means were equal to the mean value of species traits. For the analysis with environmental variables, the marginal means were computed based on residual values after accounting for all linear environmental effects and their interaction with species, included in the final model. Positive values represent species divergence in sympatry, and negative values species convergence. To allow for comparison, all traits were standardized. Stars indicate significant interactions between species and context (sympatry vs allopatry) i.e. significant pattern of convergence or divergence.

Figure 4 .

 4 Figure 4. Saturation of ventral body parts in males and females of Lissotriton helveticus (Lh) and L. vulgaris (Lv) in sympatry and allopatry. See methods for the computation of the variables. The left panel represents the raw color data for each individual and the right panel the values predicted for each individual from linear mixed models when accounting for the effects of environmental variables and species but without the effect of the context (sympatry or allopatry). The difference between the two graphs shows how sympatry change species coloration (on the left), compared to what is predicted from species-specific responses to the environmental conditions (on the right).

Dorsal coloration

S6_visible

Saturation in visible spectrum

No