Ariane Chotard
email: ariane.chotard@gmail.com

Violaine Llaurens

Vincent Debat

Patterns of morphological variation highlight the effect of natural selection on eyespots modularity in the butterfly Morpho telemachus

Keywords: Stabilizing selection, developmental constraints, variation, modularity, butterflies, fluctuating asymmetry Fluctuating asymmetry

Morphological correlations can stem from developmental constraints but also from selective pressures. Butterfly eyespots are repeated wing color pattern elements, widespread across species. As developmental serial homologues, they are controlled by similar developmental pathways imposing correlations among eyespots: selection on a single eyespot may induce correlated responses in all eyespots. We study the variations in the ventral eyespots of Morpho telemachus, where two different selective regimes are likely to act: while most eyespots are always-visible, two eyespots are conditionally-displayed: hidden at rest, they can be exposed when the butterflies are threatened, or during sexual interactions. We investigate how such contrasted selection across eyespots can alter the covariations imposed by their shared developmental origin. We quantified eyespots co-variations within a large population of M. telemachus and compared the observed patterns to those found in M. helenor, where all eyespots are always-visible and thus probably affected by a similar selection regime. We found that M. telemachus conditionally-displayed eyespots are less variable than always-visible eyespots and that these two eyespots form a separate variational module in this species, in contrast to M. helenor. Our results suggest that eyespots covariations were shaped by selection, highlighting how natural selection may promote the evolution of modularity.

Introduction

The level of evolutionary constraints on any morphological trait depends on its correlations with other traits, that originate from genetic, developmental and physical connections among traits. Ancestral correlations can strongly influence the evolution of traits under selection. For example, adaptive radiations have been suggested to occur primarily along the lines of least resistance, drawn by constraining effects of genetic correlations [START_REF] Schluter | Adaptive radiation along genetic lines of least resistance[END_REF]. But the correlations among traits can also evolve in response to selection: for example, deleterious pleiotropic effects can be eliminated or compensated for (e.g. [START_REF] Pavlicev | A model of developmental evolution: selection, pleiotropy and compensation[END_REF], and correlational selection (joint selection of several traits) can favor certain genetic correlations (e.g. [START_REF] Sinervo | Correlational selection and the evolution of genomic architecture[END_REF]. Correlations among traitstrait integrationbut also their independence from other traitstrait modularitythus stem from both selective and developmental factors (e.g. [START_REF] Wagner | Perspective: Complex adaptations and the evolution of evolvability[END_REF][START_REF] Klingenberg | Morphological Integration and Developmental Modularity[END_REF]. Whether the patterns of modularity mostly reflect the developmental constrains exerted on the evolution of traits or the selection regime is a long-standing question (e.g. [START_REF] Cheverud | Quantitative genetics and developmental constraints on evolution by selection[END_REF]Kirschner and Gerhard 1998;Schlosser and Wagner, 2005;[START_REF] Wagner | The road to modularity[END_REF][START_REF] Klingenberg | Morphological Integration and Developmental Modularity[END_REF]Mello et al. 2016).

Serial homologues, like vertebrate teeth [START_REF] Van Valen | Serial homology: the crests and cusps of mammalian teeth[END_REF] or arthropod segments [START_REF] Emerson | The Origin of Crustacean Biramous Appendages and the Evolution of Arthropoda[END_REF], are relevant traits to identify how selection can act on morphological integration. Such homologous traits stem from the repetition of the same developmental pathway in different locations of the body [START_REF] Hall | Homology and Embryonic Development[END_REF]. Serial homologues are thus expected to present a tight covariation due to their shared developmental basis [START_REF] Young | Serial homology and the evolution of Mammalian limb covariation structure[END_REF]. In contrast, heterogeneous selection across the elements of a series might drive their divergence and break down their co-variation [START_REF] Wagner | Perspective: Complex adaptations and the evolution of evolvability[END_REF][START_REF] Melo | Directional selection can drive the evolution of modularity in complex traits[END_REF]. The patterns of covariation across serial homologues therefore reflect the prevailing effect of developmental vs. selective factors affecting trait evolution [START_REF] Beldade | Concerted evolution and developmental integration in modular butterfly wing patterns[END_REF][START_REF] Allen | The "Eyespot Module" and eyespots as modules: development, evolution, and integration of a complex phenotype[END_REF]): a tight covariation across serial homologues would indicate a prominent effect of shared developmental pathways, while a modular structuration of the series, opposing co-selected elements to others, would rather suggest a prominent role of selection.

Butterfly eyespots are a textbook example of serial homology: these circular color patterns are repeated across the wings in many butterfly species. All eyespots are formed by the expression of a common developmental cascade at different locations on the wings, as shown by developmental studies carried out in the model species Bicyclus anynana (see [START_REF] Monteiro | Origin, Development, and Evolution of Butterfly Eyespots[END_REF] for a review). Such serial developmental homology results in strong genetic correlations across eyespots: artificial selection on a particular eyespot indeed induces a correlated response on all eyespots, consistent with their developmental integration [START_REF] Monteiro | The evolutionary genetics and developmental basis of wing pattern variation in the butterfly Bicyclus anynana[END_REF][START_REF] Monteiro | Butterfly eyespots: the genetics and development of the color rings[END_REF]Beldade et al. 2002;[START_REF] Beldade | The genetics and evo-devo of butterfly wing patterns[END_REF][START_REF] Klingenberg | Developmental instability as a research tool: using patterns of fluctuating asymmetry to infer the developmental origins of morphological integration[END_REF].

The wide diversity of eyespot morphologyincluding size, shape, and colorsuggests that diverse selective pressures affect their evolution [START_REF] Kodandaramaiah | The evolutionary significance of butterfly eyespots[END_REF]. In some species, eyespots are conditionally-displayed: hidden at rest, they can be uncovered at will. Usually large and highly conspicuous, these eyespots might intimidate predators if suddenly uncovered during an attack [START_REF] Stevens | The role of eyespots as anti-predator mechanisms, principally demonstrated in the Lepidoptera[END_REF][START_REF] Dapporto | Evidence for adaptive constraints on size of marginal wing spots in the grayling butterfly, Hipparchia semele[END_REF]. Radically different eyespots observed in other species, with a small size and peripheral location, are constantly visible at rest. Such eyespots might divert predator attacks away from the vital parts of the body [START_REF] Lyytinen | Does predation maintain eyespot plasticity in Bicyclus anynana?[END_REF][START_REF] Stevens | The role of eyespots as anti-predator mechanisms, principally demonstrated in the Lepidoptera[END_REF][START_REF] Olofsson | Marginal Eyespots on Butterfly Wings Deflect Bird Attacks Under Low Light Intensities with UV Wavelengths[END_REF][START_REF] Prudic | Eyespots deflect predator attack increasing fitness and promoting the evolution of phenotypic plasticity[END_REF]. The continuum of ecological contexts encountered by different butterfly species may explain the important variations in eyespots across species [START_REF] Kodandaramaiah | The evolutionary significance of butterfly eyespots[END_REF]. On top of selection by predators, sexual selection might also affect eyespot evolution. In B. anynana, UV reflectance in the center of the ventral conditionally-displayed eyespot, affects males mate choice [START_REF] Huq | Male Bicyclus anynana Butterflies Choose Females on the Basis of Their Ventral UV-Reflective Eyespot Centers[END_REF], promoting UV coloration in these particular eyespots. The heterogeneous morphology of eyespots from the same series observed within many butterfly species suggests that contrasted selective pressures may affect them, potentially breaking their covariation and leading to their morphological divergence.

Here we investigate how contrasted selective pressures may disrupt developmental constraints, by assessing morphological covariations across eyespots within the butterfly species Morpho telemachus (Linné, 1758), where the different eyespots are likely submitted to different selective regimes. In the Morpho genus, a series of eyespots is observed on the ventral side of the wings [START_REF] Debat | What Drives the Diversification of Eyespots in Morpho Butterflies? Disentangling Developmental and Selective Constraints From Neutral Evolution[END_REF], exposed when the butterflies are resting. In contrast with most Morpho species, M. telemachus has two large conditionally-displayed eyespots on the ventral side of the forewing (Figure 1) that are usually hidden at rest by the overlapping hindwing but can be revealed when the butterfly spreads its wings. These eyespots might have an intimidating effect on predators if unmasked during an attack, as suggested in other butterfly species [START_REF] Stevens | The role of eyespots as anti-predator mechanisms, principally demonstrated in the Lepidoptera[END_REF][START_REF] Dapporto | Evidence for adaptive constraints on size of marginal wing spots in the grayling butterfly, Hipparchia semele[END_REF]: paired eyespots are indeed particularly intimidating [START_REF] Stevens | The role of eyespots as anti-predator mechanisms, principally demonstrated in the Lepidoptera[END_REF][START_REF] Inglis | The Feeding Behaviour of Starlings (Sturnus vulgaris) in the Presence of 'Eyes[END_REF]. They could also be part of a concealable sexual signal. The remaining eyespots, located on the hindwing and on the forewing tip, are alwaysvisible, suggesting they might be submitted to a different selective pressure, possibly contributing to the general cryptic appearance of the butterfly at rest. Such contrasted selective regimes across eyespots in M. telemachus provide a relevant opportunity to investigate the impact of selection on the pattern of covariation across a series of developmental homologues.

While shared developmental pathways may generate tight morphological covariations among eyespots [START_REF] Brakefield | Structure of a character and the evolution of butterfly eyespot patterns[END_REF][START_REF] Allen | The "Eyespot Module" and eyespots as modules: development, evolution, and integration of a complex phenotype[END_REF], the differential selection on a subset of eyespots should promote their relative independence.

Because the covariation between different eyespots of the series might ancestrally differ, we contrasted patterns of variations observed in M. telemachus with another Morpho species, Morpho helenor (Cramer, 1776). Like in most other Morpho species, including the two species branching at the basis of Morpho phylogeny [START_REF] Chazot | Morpho morphometrics: shared ancestry and selection drive the evolution of wing size and shape in Morpho butterflies[END_REF][START_REF] Debat | What Drives the Diversification of Eyespots in Morpho Butterflies? Disentangling Developmental and Selective Constraints From Neutral Evolution[END_REF]), all eyespots of M. helenor are always-visible, suggesting that, in contrast to M. telemachus, similar selective pressures affect all eyespots of the series. Comparing the patterns of eyespot covariation of M. telemachus and M. helenor should thus shed light on the effect of heterogeneous selection on the evolution of eyespot modularity. In M. helenor, the congruent selective and developmental effects should favor the tight covariation of all eyespots, while in M. telemachus, a pattern of covariation opposing always-visible and conditionally-displayed eyespots should be detected, would the contrasted selective pressures prevail.

We first assessed the conspicuousness of the different eyespots in the two species, by measuring the reflectance spectra of the yellow and black rings forming the eyespots and computing the color contrast perceived by avian predators. We measured the size and shape of the two rings within each eyespot and then assessed their patterns of variations and covariations at two levels. First, we assessed variance among individuals, that reflects genetic and environmental differences among individuals. Then, we study variance within individuals, i.e. fluctuating asymmetry (FA), stemming from random developmental variation leading to subtle differences between right and left sides for a given trait [START_REF] Palmer | Fluctuating Asymmetry: Measurement, Analysis, Patterns[END_REF]. FA covariation across traits is then assumed to reflect developmental integration, as it is expected to result from direct developmental interactions across traits [START_REF] Klingenberg | Developmental instability as a research tool: using patterns of fluctuating asymmetry to infer the developmental origins of morphological integration[END_REF][START_REF] Klingenberg | Morphological Integration and Developmental Modularity[END_REF][START_REF] Klingenberg | Studying morphological integration and modularity at multiple levels: concepts and analysis[END_REF].

To assess the impact of selection on trait evolution and modularity, we used two sets of analyses.

(1) By comparing the levels of variation of the different eyespots within each species, we predicted a homogeneous variation in all eyespots in M. helenor as a result of a similar selection acting on all eyespots. In contrast, if different selection pressures affect the two types of eyespots in M. telemachus, they should present contrasted levels of variation as compared to other eyespots. (2) By comparing the covariations between traits within species, we predicted a global pattern of correlation across eyespots in M. helenor, resulting from homogeneous selective effects. In contrast, in M. telemachus, we predicted different patterns of modularity in the conditionally-displayed vs. always-visible eyespots, as a result of the contrasted selective regimes.

Materials & Methods

Butterfly Samples

M. telemachus is a canopy species distributed throughout the Amazon basin, from the foothills of the Andes to the Brazilian Atlantic Forest [START_REF] Blandin | Evolution and diversification of Neotropical butterflies: insights from the biogeography and phylogeny of the genus Morpho fabricus, 1807 (Nymphalidae: Morphinae), with a review of the geodynamics of South America 24[END_REF]. We focused on an exceptional sample composed of 370 males from a single emergence bloom of M. telemachus exsusarion (Le Moult & Réal 1962) collected in 1995 in Bolivia, in the province of Chapare (Department of Cochabamba CBBA; Gilbert Lachaume, pers. com.). This sample provides a relevant opportunity to assess eyespot variability within a natural population. All individuals likely encountered similar environmental conditions, reducing the potential effects of phenotypic plasticity.

We compared the levels of variation in the different eyespots within this population of M. telemachus to the variation of the same eyespots in a closely-related species, M. helenor. We gathered specimens of M. helenor from the collections of the Muséum National d'Histoire Naturelle (Paris), by sampling 31 males originating from two localities (Chapare -Bolivia/ Perene -Peru). Since these collection specimens were originally caught by different collectors at different localities, the morphological variation measured in M. helenor combines intra-and inter-populational differenceseither genetic or environmentaland thus likely over-estimates phenotypic variation.

Estimating eyespot conspicuousness

To estimate the level of conspicuousness of the different eyespots, we measured their color contrast on a subsample of 10 individuals per species. For each eyespot, the reflectance spectrum of the yellow and the black ring was measured using a spectrophotometer (AvaSpec-ULS2048CL-EVO-RS, software AvaSoft v.8.12.0.0), sensitive to wavelengths between 200 and 1100 nm. A light source (Avalight-DH-S-BAL) covering the visible and UV wavelengths from 300 to 700 nm was used to illuminate the specimens (coupling a deuterium lamp with a spectrum of 215 to 500 nm, and a halogen lamp from 500 to 2500 nm). Our measurements were conducted while minimizing external light sources. To assess the contrast between the two rings as perceived by avian predators, we quantified chromatic and achromatic contrasts [START_REF] Olsson | Chromatic and achromatic vision: parameter choice and limitations for reliable model predictions[END_REF] using the two major visual systems documented in birds (UV and violet-sensitive respectively), as implemented in the R package PAVO2 [START_REF] Maia | PAVO 2: New tools for the spectral and spatial analysis of colour in R[END_REF]. The vision models were all applied with standard conditions (Weber fraction value of 0.05 -Dell' Aglio et al. 2018) with the following relative cone densities 0.37:0.7:0.99:1 for UV-sensitive model (UVS:S:M:L) and 0.25:0.5:1:1 for Violet-sensitive model (VS:S:M:L) [START_REF] Finkbeiner | Ultraviolet and yellow reflectance but not fluorescence is important for visual discrimination of conspecifics by Heliconius erato[END_REF]. The chromatic and achromatic contrast analyses were performed using a bootstrap procedure. Contrasts are expressed in JND (Just Noticeable Difference) with a threshold of 1 JND. Values above that threshold will be considered as noticeable by an avian observer.

Measuring eyespot size and shape: Imaging and morphometric measurements

The four wings of each individual were photographed in a photo studio under controlled LED light using a Nikon D90 (Camera lens: AF-S Micro Nikkor 60 mm 1:2.8G ED), in standardized conditions allowing to minimize shape distortion due parallax. Each eyespot is composed of two concentric rings: an external yellow ring and an internal black ring, around a central white pupil. M. telemachus usually has 9 ventral eyespots (Figure 1A-B): 4 on the forewing and 5 on the hindwing. Nevertheless, two of these eyespots (E2 and E6 -visible on Figure 1B) were very often extremely reduced or absent; in addition, these eyespots are never found in M. helenor (Figure 1B). We thus excluded them from our protocol and focused on the 7 eyespots observed on all specimens.

For both species, length and width of the yellow and black rings (respectively noted Ly, Wy and Lb, Wb, Figure 1C) were measured on all eyespots using ImageJ (version 1.8.0_112). Following previous studies [START_REF] Monteiro | Butterfly eyespots: the genetics and development of the color rings[END_REF][START_REF] Breuker | Integration of wings and their eyespots in the speckled wood butterfly Pararge aegeria[END_REF][START_REF] Allen | The "Eyespot Module" and eyespots as modules: development, evolution, and integration of a complex phenotype[END_REF], length was measured along the direction parallel to the veins framing the eyespot and passing through the center of the eyespot and width along the perpendicular to that direction (Figure 1C). To characterize the shape of the different eyespots, we computed the ratio between length and width of the two rings for each eyespot (Ry=Ly/Wy and Rb=Lb/Wb) and used (1 -R) as a measure of their departure from perfect circularity. All measurements were made on both left and right eyespots, to assess asymmetry.

Assessing patterns of variations and covariations

Estimating fluctuating asymmetry Fluctuating asymmetry (FA) is the deviation from perfect bilateral symmetry due to developmental noise, i.e. the small, random variation independently affecting the two sides of a trait during development [START_REF] Palmer | Fluctuating Asymmetry: Measurement, Analysis, Patterns[END_REF]. FA was measured as the variance of the right minus left values distribution (FA4 in the terminology of Palmer 1994). To avoid measurement bias due to the lateralization of the human observer, mirror images of the left wings were used (package TransformJ, [START_REF] Meijering | Quantitative evaluation of convolution-based methods for medical image interpolation[END_REF], and the order of measurements (right or left) randomized.

Checking measurement error and allometric effects on FA

To quantify measurement error (ME), a random sub-sample of 30 individuals was measured 3 times, and the impact of ME on FA was assessed. We estimated ME on the different traits, using the repeated measurements protocol described in [START_REF] Palmer | Fluctuating Asymmetry: Measurement, Analysis, Patterns[END_REF] andPalmer (1994). We applied two-way mixed model ANOVAs with 'side' as a fixed effect and 'individual' as a random effect, for each of the 28 variables. In these models, the interaction term (side x individual) assesses FA and its statistical significance tests whether FA is greater than ME, which is included in the residual term. For all measurements, individual variation and fluctuating asymmetry were significantly larger (on average 11.6 times) than ME, suggesting that FA is not strongly affected by error.

To test whether larger eyespots display higher asymmetries, we computed the correlation of trait asymmetry values (L-R) and trait average size (L+R)/2. As no correlation was detected (r = -0.01, p = 0.284), correction for eyespot size was not applied. In contrast, a significant positive correlation was detected between eyespot size and eyespot variance (r = 0.79, p < 0.001). Inter-individual variation of linear measurements was thus assessed by their coefficient of variation (CV). Finally, to assess whether larger butterflies tended to display higher asymmetries, we tested the correlation between asymmetry values and wing area, used as a proxy of butterfly size. A significant correlation was detected, but it was very low (r = 0.026, p < 0.01), suggesting that allometric effects are weak.

Comparing eyespots variability

We then estimated the levels of variation of the different traits within each of the two species.

Differences in mean size (Ly, Wy, Lb and Wb) and shape (ratios Ry and Rb) among eyespots were tested using pairwise Welch tests, which allows to compare means of multiple samples with unequal variances. FA and inter-individual variances were compared across eyespots using pairwise F-tests, because FA was computed as a variance. Coefficients of variation of size were compared using an asymptotic test for the equality of multiple coefficients of variation (based on the calculation of the D'AD statistic proposed by Feltz and Miller 1991 -R package "cvequality", Marwick and Krishnamoorthy 2019). Multiple testing was accounted for by using Holm-Bonferroni procedure. We then compared the intraspecific levels of variations of all eyespots between species using F-tests.

As the sample sizes were very different (370 for M. telemachus and 31 for M. helenor), the interspecific comparisons were based on a bootstrap procedure, by random subsampling 10000 batches of 31 M. telemachus.

Assessing the patterns of modularity across eyespots

To identify the different variational modules, we estimated morphological covariations between traits within each species. The covariation of morphological traits across individuals can stem from different factors, including environmental effects (plasticity), but also allelic variation in genes involved in the underlying developmental pathway (pleiotropy). In contrast, FA covariation (i.e. the covariation of trait asymmetries across individuals) results from the direct effect of shared developmental factors. Since FA is inherently random, its values can be correlated across traits only if those traits directly interact during development: this can happen when the traits are physically linked or share a part of their developmental pathways (e.g. common precursor, or global pre-patterning) [START_REF] Klingenberg | Developmental instability as a research tool: using patterns of fluctuating asymmetry to infer the developmental origins of morphological integration[END_REF][START_REF] Klingenberg | Morphological Integration and Developmental Modularity[END_REF][START_REF] Breuker | Integration of wings and their eyespots in the speckled wood butterfly Pararge aegeria[END_REF]).

We thus quantified both covariation between traits values, averaged across sides (individual covariation) and covariation between traits asymmetries (FA covariation).

We specifically compared the covariations among always-visible eyespots with those among the two conditionally-displayed eyespots of M. telemachus, by computing the average intereyespot correlations. The patterns of modularity across eyespots were estimated using correlation matrices restricted to statistically significant correlations among traits. Correlation matrices were visually displayed as networks in which each variable is a node and each correlation an edge, using the package qgraph [START_REF] Epskamp | qgraph : Network Visualizations of Relationships in Psychometric Data[END_REF]. The hypotheses of modularity were then tested using a hierarchical module partition using Ward's hierarchical cluster analysis [START_REF] Ward | Hierarchical Grouping to Optimize an Objective Function[END_REF][START_REF] Zelditch | Modularity of the rodent mandible: Integrating bones, muscles, and teeth: Modularity of the rodent mandible[END_REF] using the function pvclust of the pvclust package (Suzuki et al. 2006), which calculates p-values for hierarchical clustering via multiscale bootstrap resampling. Each node supported by a significant p-value defines a module. This method has the advantage of allowing to detect nested modularity patterns and thus, a simultaneous analysis of the intra-and inter-eyespots modularity.

All statistical analyses were carried out in R version 3.6.3 (R Core Team 2020).

Results

Contrasted levels of conspicuousness among eyespots in M. telemachus

In M. telemachus, conditionally-displayed eyespots were significantly larger than all other eyespots (41.46% for Ly, 37.10% for Wy, 49.03% for Lb and 37.67% for Wb, on average; Figure 2A, Table S1). Their shape was also significantly rounder than in other eyespots (Figure 2B, details in Tables S2). Achromatic contrasts in M. telemachus were heterogeneous across eyespots, conditionally-displayed eyespots displaying particularly high values (55.69% higher than other eyespots in UV-models and 56.81% higher in Violet-models, on average; Figure 2C).

Conversely, achromatic contrasts in M. helenor eyespots were homogeneously high, comparable to M. telemachus conditionally-displayed eyespots (Figure 2C). The reduced size and achromatic contrast of the always-visible eyespots of M. telemachus suggest a strong decrease in conspicuousness in these eyespots as compared to the conditionally-displayed eyespots of M. telemachus and to all eyespots of M. helenor. Such difference was nevertheless not observed for chromatic contrasts (Figure S1), suggesting that similar coloration was conserved throughout all eyespots in both species.

The shape of always-visible eyespots is more variable and asymmetrical in M. telemachus

For both species, the variability of eyespot size, as estimated by the variation among individuals and by fluctuating asymmetry, was quite homogeneous across eyespots: most pairwise comparisons of size variation between eyespots were non-significant (fluctuating asymmetry see Figures S2; individual variation: Ly: D'AD = 1.87, p = 0.93; Lb: D'AD = 0.91, p = 0.99; Wy: D'AD = 2.84, p = 0.83; Wb: D'AD = 2.36, p = 0.88 -Figure S3). In contrast, variability of eyespot shape was strikingly different across eyespots in M. telemachus. Overall, both individual variation and asymmetry of shape were lower in the two conditionally-displayed eyespots than in the always-visible eyespots (Figure 3), and particularly strikingly so for the yellow ring (on average 4.01 times less variable and 10.08 times less asymmetric than the always-visible eyespots). Such low variability of shape in the conditionally-displayed eyespots suggests a selective effect on the roundness of these eyespots.

Overall, eyespot shape variability tended to be lower in M. helenor than in M. telemachus: among-individual variation was on average 1.62 and 2.79 times lower, in the yellow and black rings (Fyellow= 1.62, df = 2566, p < 0.001; Fblack= 2.80, df = 2564, p < 0.001 -Figure 3), and FA was 3.13 and 2.27 times lower (Fyellow= 3.13, df = 2566, p < 0.001; Fblack= 2.27, df = 2564, p < 0.001 -Figure 3). This difference was particularly strong for always-visible eyespots, which were very variable between individuals in M. telemachus, and much more stable in M. helenor.

Combined with a generally more conspicuous appearance of all eyespots in M. helenor, this stability of eyespot shape points at a similar selection regime acting on all eyespots in this species. Overall, the comparison of the two species suggests a similarly low variability in the whole series of eyespots in M. helenor, as well as in the conditionally-displayed eyespots in M. telemachus. In contrast, the always-visible eyespots of M. telemachus are much more variable.

Conditionally-displayed eyespots form a variational module in M. telemachus

Figure 4 clearly shows that there were more correlations across traits (individual variation) than across traits asymmetries (FA). Since covariations in FA reflect direct developmental interactions among traits, correlations are expected within physically-interacting traits, e.g. within each eyespot, while correlations in FA across eyespots are expected to be limited. The patterns of covariation obtained from inter-individual variation and FA matrices were nevertheless mostly congruent. Unexpectedly, the different eyespots tended to vary independently from one another, the two rings tightly covarying within an eyespot, but more loosely among eyespots. This is particularly striking in M. helenor for individual variation (Figures 4A and5A; mean correlations: corwithin all eyespots =0.94±0.05; coramong all eyespots=0.54±0.14; see Table S3 for all correlations categories), but also for FA (Figures 4B and5B; mean correlations: corwithin all eyespots = 0.67±0.24; coramong all eyespots =0.03±0.19). Consistent with a homogeneous effect of selection on the whole series of eyespots in M. helenor, no subclustering of eyespots is detected in this species.

In contrast, in M. telemachus, the two conditionally-displayed eyespots exhibit strong covariations and clearly form a separate variational module. Their FA covariation is also three times higher than that of always-visible eyespots (corconditionally-displayed CD = 0.30±0.17; coralwaysvisible = 0.11±0.12; see Table S3 for all correlations categories). Figure 5 highlights that most eyespots form distinct modules, within which the width and length of each ring (black or yellow) are correlated. In contrast, the different measurements performed on the two conditionally-displayed eyespots tightly cluster, irrespective of eyespot identity (Figure 5C).

The same pattern is found for individual variation, the covariation among conditionallydisplayed eyespots being even almost as high as that measured within a single eyespot (corconditionally-displayed = 0.74±0.12, coralways-visible = 0.49±0.10 corwithin all= 0.82±0.18). Overall, this opposition between conditionally-displayed vs. always-visible eyespots suggests a change in the modularity pattern in M. telemachus as compared to M. helenor.

Discussion

Differential eyespot variability in M. telemachus shaped by contrasted selection regimes

In M. telemachus, the large difference in achromatic contrast detected between conditionallydisplayed eyespots and the others (Figure 2C), suggests that the two types of eyespots may be submitted to different selective regimes. Achromatic contrast is indeed often used by birds to detect small targets while chromatic contrast is involved in discrimination of large targets and recognition of chromatic patterns [START_REF] Osorio | Visual Ecology and Perception of Coloration Patterns by Domestic Chicks[END_REF][START_REF] Théry | Specific color sensitivities of prey and predator explain camouflage in different visual systems[END_REF][START_REF] Halpin | Pattern contrast influences wariness in naïve predators towards aposematic patterns[END_REF].

Achromatic contrast was also shown to increase prey conspicuousness and detection by mantid predators [START_REF] Prudic | Aposematic coloration, luminance contrast, and the benefits of conspicuousness[END_REF]. Our results thus highlight that conditionally-displayed eyespots are more conspicuous than the other eyespots. Their evolution might thus have been influenced by different selective pressures.

Our analyses then showed that in M. telemachus, conditionally-displayed eyespots are larger and rounder than the always-visible eyespots. Their shape is also strikingly less variable, with levels of variation and fluctuating asymmetry (FA) comparable to those detected in M. helenor (Figure 3). This is consistent with an effect of stabilizing selection, expected to reduce genetic variation [START_REF] Stearns | The differential genetic and environmental canalization of fitness components in Drosophila melanogaster[END_REF][START_REF] Boonekamp | Canalisation in the wild: effects of developmental conditions on physiological traits are inversely linked to their association with fitness[END_REF]. Stabilizing selection might also favor eyespot developmental robustness, reducing variation and FA via enhanced canalization and developmental stability [START_REF] Schmalhausen | Factors of evolution: the theory of stabilizing selection[END_REF][START_REF] Palmer | Fluctuating Asymmetry: Measurement, Analysis, Patterns[END_REF][START_REF] Clarke | The genetic basis of developmental stability. V. Inter-and intra-individual character variation[END_REF][START_REF] Leamy | The Genetics and Evolution of Fluctuating Asymmetry[END_REF][START_REF] Garnier | Hybridization, developmental stability, and functionality of morphological traits in the ground beetle Carabus solieri (Coleoptera, Carabidae)[END_REF]but see Pelabon et al. 2010).

Most Morpho species, including the species M. eugenia and M. marcus, branching at the basis of the Morpho phylogeny, display large and conspicuous eyespots similar to those of M. helenor [START_REF] Debat | What Drives the Diversification of Eyespots in Morpho Butterflies? Disentangling Developmental and Selective Constraints From Neutral Evolution[END_REF]. The small size and high variability of the always-visible eyespots in M. telemachus is thus likely a derived condition. This higher variability might be related to a relaxation of selection on these eyespots. Non-functional and vestigial structures are indeed expected to present increased morphological variance and FA (reviewed in [START_REF] Lahti | Relaxed selection in the wild[END_REF], as observed in the reduced wings of some insects (e.g. carabid: [START_REF] Garnier | Hybridization, developmental stability, and functionality of morphological traits in the ground beetle Carabus solieri (Coleoptera, Carabidae)[END_REF]gall thrips: Crespi and Vanderkist, 1997) or the reduced digits of some mammals (in primates: [START_REF] Tague | Variability of a vestigial structure: first metacarpal in Colobus guereza and Ateles geoffroyi[END_REF][START_REF] Tague | Variability of metapodials in primates with rudimentary digits: Ateles geoffroyi, Colobus guereza, and Perodicticus potto[END_REF], or canids: Tague, 2020). We recently reported a negative correlation between eyespot size and intraspecific variability of eyespot size and shape across the whole Morpho genus [START_REF] Debat | What Drives the Diversification of Eyespots in Morpho Butterflies? Disentangling Developmental and Selective Constraints From Neutral Evolution[END_REF], suggesting that beyond the particular example of M. telemachus, stabilizing selection on the smallest eyespots might be relaxed compared to larger ones.

The high variability of M. telemachus always-visible eyespots might also be explained by other specific selective regimes, like apostatic selection [START_REF] Allen | Evidence for Apostatic Selection by Wild Passerines[END_REF]Ursprung andNöthiger 1972 Bond 2007), a form of negative frequency-dependent selection favoring rare phenotypes in prey. Rarely encountered morphologies would be more difficult to identify by predators, as suggested for the cryptic patterns of moths [START_REF] Bond | The Evolution of Color Polymorphism: Crypticity, Searching Images, and Apostatic Selection[END_REF]Kamil 2002, 2006).

Apostatic selection may thus favor low frequency variants in M. telemachus always-visible eyespots, improving crypsis. While this selection in principle relies on genetic variants, it might also favor low levels of developmental robustness. The high FA measured in these eyespots raises the interesting possibility of an adaptive developmental instability [START_REF] Forde | Is it good noise? The role of developmental instability in the shaping of a root system[END_REF].

The pattern of eyespot modularity is likely shaped by selection in M. telemachus

In both species, the covariation among eyespots was markedly lower than within eyespots, for both FA and individual variation. This result was expected for FA, as direct developmental interactions across eyespots are expected to be limited to the common global pre-patterning of the wing or to physically close traits (e.g. [START_REF] Breuker | Integration of wings and their eyespots in the speckled wood butterfly Pararge aegeria[END_REF]). Considering individual variation, the relative independence of the different eyespots was unexpected. Eyespots serial homology indeed implies that genetic variation affecting any of the components of the shared genetic network should trigger a joint phenotypic variation. Similarly, any environmental influence on this common network should increase phenotypic covariance (e.g. [START_REF] Allen | The "Eyespot Module" and eyespots as modules: development, evolution, and integration of a complex phenotype[END_REF]. A tight covariation across eyespots was in particular predicted in M. helenor, where all eyespots are expected to be submitted to the same selection regime. The relative independence of the different eyespots thus suggests that their formation involves locally different processes, allowing some independent variation. This result is consistent with artificial selection experiments in B. anynana (Beldade et al. 2002;[START_REF] Beldade | Concerted evolution and developmental integration in modular butterfly wing patterns[END_REF], showing that the independent evolution of eyespots is not strongly constrained by the genetic correlations among eyespots.

A remarkable exception to this global pattern of independence is the tight covariation between the two conditionally-displayed eyespots of M. telemachus. In particular, their covariance in FA suggests that specific developmental processes have evolved that jointly affect these two eyespots but not the others. This change in modularity, opposing the conditionally-displayed eyespots to the always visible eyespots, is likely shaped by the contrasted selective pressures affecting them. Such a change in modularity may result from the adaptive suppression of pleiotropy across groups of traits submitted to different selective pressures [START_REF] Cheverud | Quantitative genetics and developmental constraints on evolution by selection[END_REF][START_REF] Wagner | Perspective: Complex adaptations and the evolution of evolvability[END_REF][START_REF] Breuker | Functional evo-devo[END_REF][START_REF] Wagner | The road to modularity[END_REF][START_REF] Klingenberg | Studying morphological integration and modularity at multiple levels: concepts and analysis[END_REF].

Studies investigating the adaptive evolution of modularity are still largely lacking [START_REF] Breuker | Functional evo-devo[END_REF][START_REF] Klingenberg | Quantitative genetics of shape in cricket wings: developmental integration in a functional structure[END_REF]. Our study on the eyespots of M. telemachus may thus represent a relevant case where modularity can be tuned by natural selection [START_REF] Breuker | Functional evo-devo[END_REF]. Similar analysis of modularity should be performed in other species harboring heterogeneous morphologies of eyespots, to assess how selection can affect developmental covariations between traits. Experiments are now required to identify (1) the exact selection regime affecting conditionally-displayed eyespots in M. telemachus and, (2) the developmental bases underlying the evolution of modularity across Morpho species. The height of the nodes indicates the distance between two observations (here we used correlation matrix as distance matrix, so the higher the height, the less correlated are two traits).

Figures legends

A node market with a blue circle indicate that the associated cluster is significant. The cluster associating the conditionally-displayed eyespots E3 and E4 in M. telemachus is the only cluster whose intra-eyespot modularity is overcome by inter-eyespots modularity.

Appendices

Figure 1 :

 1 Figure 1: Eyespots observed on the ventral sides of the wings in Morpho telemachus and Morpho helenor. (A) Picture of M. telemachus taken in resting position, showing the alwaysvisible eyespots. Photo credit: Peter Møllmann. (B) Position and numbering of the measured eyespots. Eyespots E3 and E4, figured in dark and light orange, are conditionally-displayed in M. telemachus (they are usually hidden by the hindwing at rest), and always exposed in M. helenor. The other eyespots are figured in grey. Left -M. telemachus: Right -M. helenor. (C)The four measurements taken on each eyespot (length and width of the yellow and black rings, respectively noted Ly, Wy, Lb and Wb).

Figure 2 :

 2 Figure 2: Sizes, shapes and colors of eyespots, revealing contrasted levels of conspicuousness in M. telemachus and M. helenor. Eyespots E3 and E4 (conditionallydisplayed in M. telemachus) are figured in orange. Circles: M. telemachus; triangles: M. helenor. A: Eyespots sizes. The four measurements are similarly different across eyespots, so only Ly is displayed. Boxplots indicate mean and standard deviation. Significant differences between eyespots are shown using different letters (a, b, c)results for M. telemachus are

Figure 3 :

 3 Figure 3: High levels of shape variability in M. telemachus always-visible eyespots suggest a relaxed selection. Inter-individual variation (top) and fluctuating asymmetry (bottom) of eyespots yellow ring shape (Ly/Wy). For each species, significant differences between eyespots are shown using different letters (a, b, c; a letter pools non-significantly different traits). Left: M. telemachus (n = 370); right: M. helenor (n = 31). The relatively low variability of E3 and E4 is comparable to that observed in most M. helenor eyespots, suggesting a similar stabilizing selection. The high variability of M. telemachus always-visible eyespots in turn suggests a relaxed or apostatic selection.

Figure 4 :

 4 Figure 4: Contrasted patterns of modularity observed in M. telemachus and M. helenor. as assessed by correlation matrices of linear parameters (Ly, Wy, Lb, Wb) of the 7 different eyespots. Top: M. helenor (n = 31); Bottom: M. telemachus (n = 370). Left: modularity patterns inferred from individual variation; Right: modularity patterns inferred from FA. Nodes represent the 4 measured variables and the edges represent the statistically significant correlations. Line thickness is proportional to the correlation. The blue ellipse shows the module regrouping E3 and E4 detected by the hierarchical clustering in M. telemachus.

Figure 5 :

 5 Figure 5: Hierarchical clustering based on the FA and inter-individual correlation matrices of linear parameters (Ly, Wy, Lb, Wb) of the 7 different eyespots. M. telemachus (n = 370) is shown on the second row and M. helenor (n = 31) on the first row. Left: Hierarchical clustering based on the inter-individual correlation matrices. Right: Hierarchical clustering based on FA correlation matrices. Hierarchical clustering exploring the networks modularity.

Figure S1 :

 S1 Figure S1: Chromatic contrasts (JND) between the yellow ring and the black ring of eyespots measured in M. telemachus (n = 10; circles) and M. helenor (n = 10; triangles). The 2 vision models are displayed: UV-sensitive birds (left) and Violet-sensitive birds (right).

Figure S2 :

 S2 Figure S2: Fluctuating asymmetry of eyespots linear parameters, respectively length and width of the yellow ring (Ly and Wy) and length and width of the black ring (Lb and Wb) of each eyespot (E1, E3, E4, E5, E7, E8 and E9), measured in the M. telemachus sample (n = 370) and M. helenor (n = 31). Significant differences between eyespots are shown using different letters (a, b, c)results for M. telemachus are displayed in full letters (above) and results for M. helenor are displayed in italic letters (below).

Figure S3 :

 S3 Figure S3: Inter-individual variation of eyespots linear parameters, respectively length and width of the yellow ring (Ly and Wy) and length and width of the black ring (Lb and Wb) of each eyespot (E1, E3, E4, E5, E7, E8 and E9), measured in the M. telemachus sample (n = 370) and M. helenor (n = 31).

  E5 E7 E8 E9 E1 E3 E4 E5 E7 E8 E9 E1 E3 E4 E5 E7 E8 E9