Melanie Mcclure
email: mel_mcclure@hotmail.com

Corentin Clerc

Charlotte Desbois

Aimilia Meichanetzoglou

Marion Cau

Lucie Bastin-Héline

Javier Bacigalupo

Céline Houssin

Charline Pinna

Bastien Nay

V Llaurens

S Berthier

C Andraud

D Gomez

Why has transparency evolved in aposematic butterflies? Insights from the largest radiation of aposematic butterflies, the Ithomiini

Keywords: crypsis, conspicuousness, detectability, honest signal, Müllerian mimicry, unpalatability

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Organisms utilize a range of defensive strategies that are effective in reducing the probability of being attacked by predators [1]. These include avoiding detection, such as camouflage or crypsis, or deterring predators from attacking when encountered, such as aposematism, whereby defended prey advertise their unpalatability to predators using conspicuous warning displays [see 2].

Aposematism relies on the fact that predators learn to associate certain features that act as warning signals (such as coloration, sounds, odors) with distastefulness upon attacking defended prey.

There is evidence that predators learn to avoid unpalatable prey more quickly and remember the association between the signal and unpalatability for longer and make fewer recognition errors if prey are more conspicuous [3][4][5]. The benefits of being conspicuous are also thought to include producing stronger neophobic avoidance by naïve predators, a greater reluctance to attack and a lower probability of killing an aposematic prey in an attack as predators may engage in cautious sampling and/or taste rejection of unpalatable prey [6,7].

Hence, defended species often have conspicuous signals that warn potential predators of these defenses. Because predators that attack aposematic individuals learn to avoid prey harboring a specific signal, aposematic signals are under positive frequency-dependent selection [8,9], whereby the efficiency of a signal increases with abundance. Unpalatable species exposed to the same suite of predators therefore tend to converge on the same warning signals because these individuals effectively share the cost of educating predators, and this is known as Müllerian mimicry [START_REF] Müller | Ituna and Thyridia; a remarkable case of mimicry in butterflies[END_REF].

Aposematism and Müllerian mimicry occur in a range of organisms, but it has been especially well studied in butterflies [START_REF] Sherratt | The evolution of Müllerian mimicry[END_REF]. The Neotropical butterfly tribe Ithomiini (Nymphalidae: Danainae) comprises ca. 393 species and 53 genera, and is the largest known radiation of mimetic butterflies. Ithomiini numerically dominate forest butterfly communities and are thought to drive convergence in wing color pattern in multiple mimicry rings [START_REF] Brown | Adaptive polymorphism associated with multiple Müllerian mimicry in Heliconius numata (Lepid. Nymph.)[END_REF][START_REF] Beccaloni | Ecology, natural history and behaviour of ithomiine butterflies and their mimics in Ecuador (Lepidoptera: Nymphalidae: Ithomiinae)[END_REF], whereby co-occurring aposematic species, of both Ithomiini and other Lepidoptera lineages, converge in their aposematic signal. Although several species are brightly colored, over 80% of Ithomiini have more subdued color patterns as a result of transparency [START_REF] Beccaloni | Ecology, natural history and behaviour of ithomiine butterflies and their mimics in Ecuador (Lepidoptera: Nymphalidae: Ithomiinae)[END_REF][START_REF] Mallet | Individual selection, kin selection, and the shifting balance in the evolution of warning colours: the evidence from butterflies[END_REF], and as such, the tribe is commonly known as clearwing butterflies. Transparency is likely a derived state, since many basal Ithomiini lineages and most species of the sister clade Danaini are conspicuously colored and not transparent.

Nevertheless, all species of Ithomiini, even those that are transparent, exhibit distinct and recognizable color patterns and, like other unpalatable butterflies, are similarly colored on both sides of their wings and are characterized by slow flight (often described as part of a chemical defense syndrome). Some species have been shown to be highly unpalatable to both vertebrate and invertebrate predators [START_REF] Brown | Adult-obtained pyrrolizidine alkaloids defend ithomiine butterflies against a spider predator[END_REF][START_REF] Brower | Birds, butterflies and plant poisons: a study in ecological chemistry[END_REF][START_REF] Chai | Field observations and feeding experiments on the responses of rufous-tailed jacamars (Galbula ruficauda) to free-flying butterflies in a tropical rainforest[END_REF] as a result of the pyrrolizidine alkaloids (PAs) they contain. Like Danaini butterflies (sister-group to the Ithomiini) and arctiid moths, some basal Ithomiini species sequester PAs from larval food plants (Apocynaceae), whereas other Ithomiini that have shifted to Solanaceae or Gesneriaceae as hostplants obtain PA compounds as adults by feeding on flowers of Asteraceae and Boraginaceae [START_REF] Pliske | Attraction of Lepidoptera to plants containing pyrrolizidine alkaloids[END_REF][START_REF] Devries | Attraction of Pyrrolizidine Alkaloid seeking Lepidoptera to Epidendrum paniculatum orchids[END_REF][START_REF] Trigo | Variation of pyrrolizidine alkaloids in Ithomiinae: a comparative study between species feeding on Apocynaceae and Solanaceae[END_REF].

As such, the evolution of transparency in Ithominii butterflies is very puzzling.

Transparency is generally assumed to be involved in concealment, but this has mostly been studied in aquatic systems where it is abundant [START_REF] Johnsen | Hidden in plain sight: the ecology and physiology of organismal transparency[END_REF][START_REF] Johnsen | Hide and seek in the open sea: pelagic camouflage and visual countermeasures[END_REF]. If transparency has indeed evolved as a result of selection for concealment, these butterflies may benefit from both crypsis and aposematism [START_REF] Barnett | Distance-dependent defensive coloration in the poison frog Dendrobates tinctorius, Dendrobatidae[END_REF][START_REF] Tullberg | Aposematism and crypsis combined as a result of distance dependence: functional versatility of the colour pattern in the swallowtail butterfly larva[END_REF][START_REF] Kang | Body size affects the evolution of hidden colour signals in moths[END_REF]. However, because conspicuousness of unprofitable prey is thought to act as an honest indicator of toxicity (see [START_REF] Maan | Poison frog colours are honest signals of toxicity, particularly for bird predators[END_REF][START_REF] Cortesi | Conspicuousness is correlated with toxicity in marine opisthobranchs[END_REF][START_REF] Sherratt | The evolution of warning signals as reliable indicators of prey defense[END_REF][START_REF] Blount | Warning displays may function as honest signals of toxicity[END_REF] but see also [START_REF] Wang | Inversely related aposematic traits: reduced conspicuousness evolves with increased toxicity in a polymorphic poison-dart frog[END_REF][START_REF] Darst | A mechanism for diversity in warning signals: Conspicuousness versus toxicity in poison frogs[END_REF]) and predators have been shown to preferentially avoid brighter prey [3], unprofitable species that are inconspicuous are generally expected to possess weaker defenses.

Here we test the hypotheses that transparent Ithomiini species are both (1) less detectable by predators and (2) less unpalatable as a result. We estimate detectability by natural predators (i.e. birds) of 33 species of Ithomiini butterflies spanning a range of transparency, from fully opaque to largely transparent and unpigmented, by measuring transmittance and reflectance of the wings, and using models of avian vision. We then assess the unpalatability of 10 species by measuring the PA content and by recording taste-rejection of avian predators (domestic chicks). S1 for a list of species and number of individuals used) collected for optical (N=33 species; see Figure S1 for photographs of the taxa used) and chemical analysis (N=10 species) were preserved dry in envelopes until use. Specimens to be used to measure unpalatability using chicks (N=10 species) were kept in ambient conditions in 2 x 4 x 2 m outdoor insectaries until use. Butterflies were used no more than a few days after capture so as to ensure that they were fresh and reflected naturally occurring toxin content.

Methods

Specimens used

Butterflies

Specimens (see Table

Optical measurements, transparency and detectability

To assess the relationship between detectability and transparency, optical measurements were performed on 33 Ithomiini species. Although none of the species harbor obvious sexual dimorphism in wing pattern, all individuals used were female when possible so as to avoid any sex-related differences (see Table S1 for details). Transparent species typically possess large transparent areas that can harbor different colors (e.g., yellow, orange, white or none), in addition to opaque areas of varying sizes and colors (Figure S1).

Using spectrometry (see Supplementary Methods for details), the transmittance spectra of the transparent areas and the transmittance and reflectance spectra of the opaque areas were measured. The average of the measurements for transmittance (five on the forewing and five on the hindwing) taken in different transparent areas of the wing was used to assess transmittance of transparent patches and account for the heterogeneous properties across transparent areas.

Differences between individuals of the same species was found to be minimal and less than that found between species (see Supplementary Methods for details). Therefore, one individual per species was used in subsequent analyses, except for those species for which two individuals were measured and for which we used the mean of both individuals. For each species a transparency index was computed as the average of the transmittance of transparent and opaque areas in the fore-and hindwing weighted by the proportion of surface occupied by those areas. This index, which can vary between 0 (fully opaque) and 100 (fully transparent), therefore takes into account both the proportion of transparent patches in the wing, and the degree of transparency of those patches. Butterfly size was not taken into account in this index.

The brightness and color contrast of butterflies against a background of vegetation as perceived by birds was quantified using visual modelling and Vorobyev & Osorio's discriminability model [START_REF] Vorobyev | Receptor noise as a determinant of colour thresholds[END_REF]. Given that spectral sensitivities of the avian predator community in this study system are unknown, calculations were based on the two main vision systems found in birds: ultraviolet sensitive (UVS) and violet-sensitive (VS) vision (see details of model parameters in SI). Light conditions used for the vision models simulated ambient light conditions in forest shade, common in rainforest understory, and the reflectance spectrum of an average tropical leaf as a visual background [START_REF] Gomez | Simultaneous crypsis and conspicuousness in color patterns: comparative analysis of a neotropical rainforest bird community[END_REF]. For transparent areas of the wing, the ambient light was assumed to be transmitted by the wing, reflected by the leaf and transmitted again by the wing. For opaque areas, the ambient light was assumed to only be reflected by the wing before reaching the observer.

For the semi-transparent species Ceratinia tutia and C. neso, measures of reflectance rather than transmittance were used because these species had very low transmittance in any given transparent areas. The color and brightness contrast of the wings were obtained by averaging the contrast measured for each colorful or transparent area weighted by the surface area. All computations were done using the Pavo R package [START_REF] Eliason | pavo: an R package for the analysis, visualization and organization of spectral data[END_REF].

Because species are not independent due to shared ancestry, the relationship between transparency and detectability was assessed by performing phylogenetic regressions of our detectability estimates against the transparency index using the R package caper [START_REF] Orme | Caper: comparative analyses of phylogenetics and evolution in R. R package[END_REF]. We extracted the phylogeny of our 33 species from the Ithomiini phylogeny [START_REF] Chazot | Renewed diversification following Miocene landscape turnover in a Neotropical butterfly radiation[END_REF], which contains 340 species, including all those used in this study. For each estimate of detectability, the phylogenetic signal of the residuals of the regression (Pagel's lambda) was estimated and the phylogeny was rescaled accordingly, such that only the amount of phylogenetic signal in the actual data was accounted for [START_REF] Symonds | A primer on phylogenetic generalised least squares[END_REF].

Experiments measuring unpalatability using domestic chicks

Domestic chicks (Gallus domesticus) of ca. 10 days old were used as model predators (see Supplementary Methods for further details of the methodology described below; see also [START_REF] Chouteau | Natural prey with diverse defense levels engage in mutualistic mimicry[END_REF][START_REF] Skelhorn | Prey palatability influences predator learning and memory[END_REF][START_REF] Gamberale-Stille | Automimicry destabilizes aposematism: predator sample-and-reject behaviour may provide a solution[END_REF]) to test the unpalatability of 10 different transparent or conspicuously colored Ithomiini butterfly species (see Figure 1). Prey consisted of orange or green colored pellets of chick feed, either with or without freshly killed butterflies mixed into the paste (experimental vs control pellets; see Supplementary Methods for details). A total of six chicks were tested for each butterfly species. Birds had no prior experience of unpalatable prey and were initially trained to eat the dyed chick feed. Chicks were tested with all palatable pellets of both colors before the start of the experiment, and only those that showed no bias were used. Chicks were then presented with a plastic tray containing 20 pellets of each color, presented alternatingly, for a total of 40 pellets (i.e. the chicks were given a choicetest with 40 pellets, half of which were of one color and palatable, and the other half another color and unpalatable). Chicks were allowed to attack (peck or eat) 20 pellets before the food tray was removed. These chicks were then tested for a total of 12 successive trials, always with the same color combination. By reversing the color association for half the birds that were tested (i.e. three chicks were tested with green experimental pellets and three chicks were tested with orange experimental pellets, for a total of six chicks tested for each butterfly species), we ensured that there was no color bias in the pellets that were preferentially attacked. In each session the number of palatable and unpalatable pellets attacked were recorded. Intra-individual variation in the response of chicks to a given butterfly species was found to be minimal (see Supplementary Methods).

To evaluate unpalatability of the different butterfly species, the following exponential decrease model was fitted to the data pooled for all six chicks (see Figure S2) using the nls function in R:

10 .
where t is the trial number, E(t) the number of experimental pellets eaten at trial t, and A and T are constants. This is because the shape of avoidance learning curves, whereby predators reduce their attack rates on defended prey over a series of test sessions, has been shown to occur in two phases [START_REF] Skelhorn | Learning about aposematic prey[END_REF]. The first is an acquisition phase, which shows the speed with which a predator learns to associate the warning signal with the defense, and is depicted by the half-life of the exponential decrease model. This is illustrated in the above equation by the variable T, and the higher the value, the more trials are necessary for the chicks to learn to avoid the unpalatable pellets. The second is an asymptotic phase, illustrated by the variable A, where predators appear to have learned the association and do not change their attack rate. A and T therefore decrease with increasing unpalatability. Fully palatable pellets should lead to infinite values of T, which cannot be analyzed subsequently, and we therefore arbitrarily set the upper limit to 100.

In the above equation, discretization of the time variable t, which is the number of trials, was accomplished by adding an offset of -0.5. This was done because avoidance learning does not take place at the end of trials, but during the trials. Consequently, the number of pellets recorded as attacked by the chicks during a given trial t actually occurs over time between t-1 and t. The start of each trial can therefore be set to t-0.5 if the attack rate is assumed to be linear between these two points. Changes in attack rates are especially fast during the first few trials and the model parameters are therefore very sensitive to the offset. Finally, the rate of attack by chicks was set to 10 before the start of the first trial so as to indicate that chicks were feeding on both colors without bias, as was tested at the start of each experiment. In the equation, this is done by setting the yintercept (A+(10-A)) to 10 before the start of the first trial (i.e. for E(0.5)).

To test the relationship between unpalatability (estimated by both the above variables A and T) and estimates of detectability, a phylogenetic regression of detectability on unpalatability was performed as detailed in the previous section (correcting for the exact amount of phylogenetic signal), using the R package caper [START_REF] Orme | Caper: comparative analyses of phylogenetics and evolution in R. R package[END_REF]. In addition, we tested whether transparent species as a group (transparency index >20%; see Figure 1) differed from opaque species as a group in their unpalatability by performing a phylogenetic ANOVA using the R package phytools [START_REF] Revell | phytools: an R package for phylogenetic comparative biology (and other things)[END_REF], and implementing 1000 simulations.

Chemical quantification of pyrrolizidine alkaloids (PAs)

PA content (free bases and N-oxides) of the 10 species tested with the chick behavioral assays above was quantitatively analyzed by colorimetric assay [START_REF] Trigo | Pyrrolizidine alkaloids in the arctiid moth Hyalurga syma[END_REF]. Individual butterflies (body and wings) were weighed and pulverized using liquid nitrogen, and placed into vials containing 2 mL MeOH. These vials were then stirred continuously for 3 h at room temperature. After filtration using glass pipettes and cotton, 100 µL of the liquid extract was mixed with 0.5 mL of oxidation reagent (5 mg of Na4P2O7 in 1 mL of H2O2 30%, diluted 1:200 with MeOH) and then heated in open test tubes at 100°C for 25 min, until complete evaporation of the solution. The dried samples were left to cool to room temperature, and placed overnight in a desiccator so as to remove any traces of residual water. Anhydrous isoamyl acetate (1.0 mL) and acetic anhydride (0.1 mL) were then added to the tubes, covered so as to avoid any water contamination, and placed at 100°C for 2 min. Samples were again cooled to room temperature and treated with 1.0 mL of freshly prepared modified Ehrlich reagent (8 mL of 20% methanolic boron trifluoride (BF3), diluted in 72 mL of absolute EtOH and 1.4 g dimethylaminobenzaldehyde). Tubes were sealed and heated at 60°C for 5 min and cooled to room temperature for 23 minutes. Samples were assayed photometrically at 561 nm against a blank solution (a mixture of isoamyl acetate, acetic anhydride and modified Ehrlich reagent). An initial solution of 5 mg of crotaline and 5 mg of retrorsine dissolved in 10 mL of MeOH (initial concentration: 1 mg/mL) was used to produce six different dilutions in MeOH, which were then used to produce a reference curve.

Average PA content (per unit of weight for each butterfly) was compared between species and between sexes using a non-parametric Kruskal-Wallis test done using SPSS (SPSS Inc., Chicago, Illinois). A phylogenetic regression (as above) was done to test the correlation between PA content and unpalatability, using both the variables A and T calculated from the above avoidance curves. Finally, the relationship between PA content and 1) detectability and 2) transparency were assessed by performing phylogenetic regressions and a phylogenetic ANOVA respectively, as described in the previous section.

Table 1 shows average transparency, detectability, unpalatability and toxin content for all 10 Ithomiini species tested.

Results

Transparency and detectability

The relationship between transparency (as measured by a transparency index that takes into account the proportion of the different color patches and their respective transparencies) and detectability by predators was assessed by measuring the transmittance (the amount of light that passes through the wings) and the reflectance (the amount of light that is reflected) spectra of wings using spectrometry and models of avian vision. The 33 species used in this study span all major clades of Ithomiini (Figure S3) and a wide range of transparency, ranging from completely opaque, with a transparency index of zero (e.g. Melinaea mothone, Mechanitis messenoides, Hypothyris mansuetus), to highly transparent (e.g. Heterosais nephele, Ithomia agnosia, Pteronymia gertschi) (Figure S1). Detectability (estimated by the chromatic and achromatic contrasts of wing patterns against a green leaf) decreased with increasing butterfly transmittance, regardless of vision type (UVS or VS), type of contrast (chromatic or achromatic), or simulated light conditions (Table 2, Figure 2).

Detectability and unpalatability

The unpalatability of 10 of the previous 33 Ithomiini species (Figure 1) that differ in their degree of transparency was tested using domestic chicks. Two facets of unpalatability were assessed in our analyses (Figure S2): the speed of avoidance learning, depicted by variable T, and the number of residual attacks sustained by educated predators, depicted by variable A. This last variable can also be thought of as the strength and durability of avoidance learning. Both A and T decrease with unpalatability (i.e., the more unpalatable a species, the more quickly predators learn to avoid them (T) and the stronger the avoidance over time (A)).

The relationship between unpalatability and detectability was first ascertained with a phylogenetic regression. In this analysis, neither the speed of avoidance learning T (Table 3, Figure 3a), nor the number of attacks sustained by educated predators A (Table 4, Figure 3b) were significantly associated with detectability of butterflies, across both types of predators, both types of contrasts and both types of simulated light conditions, despite a trend for a positive relationship between chromatic contrast and variable A. However, when transparency was considered as a categorical variable (i.e., largely transparent or opaque), transparent species were found to be significantly more unpalatable when considering the number of attacks sustained by educated predators, as estimated by variable A (transparent: A=0.469±0.468; opaque: A=4.653±4.496; phylogenetic ANOVA F=5.446, df=1, p=0.044; Figure 3b), but not the speed of avoidance learning, as estimated by variable T (transparent: T=2.305±2.670; opaque: T=26.446±49.038; phylogenetic ANOVA F=5.446, df=1, p=0.245; Figure 3a). A single Ithomiini species, the colorful and weakly transparent Ceratinia tutia (considered as opaque, see Supplementary Methods), was found to be fully palatable to chicks.

Chemical quantification of pyrrolizidine alkaloids (PAs), and comparison with unpalatability and detectability

PA content (μg/mg) of the 10 species tested with the chick behavioral assays was quantitatively analyzed by colorimetric assay and was found to be significantly different between species (Wald=62.877; df=9; p<0.001; Figure S4) but not between males and females (Wald=2.021; df=1; p=0.155; Figure S4), although content appears to be more variable within males. Surprisingly, no PAs were detected in any individual of the species Methona confusa. PA content was also low for the species Tithorea harmonia, known to sequester PAs from their hostplants as larvae [START_REF] Trigo | Variation of pyrrolizidine alkaloids in Ithomiinae: a comparative study between species feeding on Apocynaceae and Solanaceae[END_REF]. Surprisingly, PA content did not predict unpalatability and there was no relationship between PA content and the speed of avoidance learning T (Table S2a; Figure S5a) nor the number of attacks sustained by educated predators A (Table S2b; Figure S5b). For example, Methona confusa, which contained little to no PAs, were found to be highly unpalatable to chicks, and Ceratinia tutia, which possessed average quantities of PAs, were found to be palatable to chicks.

There was no significant relationship between PA content and detectability (Table S3).

When transparency was considered as a categorical variable (i.e., largely transparent or opaque), there was no significant difference in PA content between groups (free PA content of transparent species=19.2±9.8 and of opaque species=17.8±10.1; phylogenetic ANOVA F=0.0496, df=1, p=0.836. N-oxides PA content of transparent species=18.6±9.5 and of opaque species=17.5±10.0; phylogenetic ANOVA F=0.0378, df=1, p=0.850).

Discussion

Transparency has likely evolved in clearwing butterflies (Ithomiini) from a brightly colored aposematic ancestor, and this study shows that it has resulted in a significant decrease in detectability by avian predators (i.e. improved concealment). This is consistent with results from four Ithomiini species that showed that transparency decreases detectability for both human observers and blue tits [START_REF] Arias | in press Transparency reduces predator detection in chemically protected clearwing butterflies[END_REF]. Surprisingly, we did not find a concurrent decrease in unpalatability with increasing transparency. Despite considerable empirical support that conspicuousness promotes the effectiveness of aposematic signals, and thus that selection should favor conspicuous signals in well-defended individuals, there are many examples of unprofitable species that are rather cryptic [START_REF] Wang | Inversely related aposematic traits: reduced conspicuousness evolves with increased toxicity in a polymorphic poison-dart frog[END_REF][START_REF] Lindstedt | Disentangling the evolution of weak warning signals: high detection risk and low production costs of chemical defences in gregarious pine sawfly larvae[END_REF].

Various reasons have been put forward to explain weak aposematic signals. Some studies have shown that in addition to greater conspicuousness, greater toxicity of prey can also increase the speed and efficiency of predator avoidance learning [9,[START_REF] Skelhorn | Prey palatability influences predator learning and memory[END_REF], and this could potentially reduce selection on brightly colored visual patterns [START_REF] Wang | Inversely related aposematic traits: reduced conspicuousness evolves with increased toxicity in a polymorphic poison-dart frog[END_REF]. Here we found that the most transparent species were all highly unpalatable. In fact, transparent species were found to be significantly more unpalatable than opaque species, the latter spanning a larger range of unpalatability, including one fully palatable species. Indeed, although the speed of avoidance learning (T) was not significantly different for both transparent and conspicuously colored species, the number of residual attacks sustained by educated predators (A) was significantly reduced for transparent species in the absence of a visual cue. This suggests that some aspects of taste, rather than conspicuousness, may improve the memorability of unpalatability in transparent species. However, it should be noted that as a result of the relatively small number of species tested, the difference in unpalatability between transparent and opaque species may not be accurately estimated. This does not refute the findings that all transparent species tested are highly unpalatable, but it may be premature to conclude that transparent species are systematically more unpalatable than conspicuous species.

Reduced conspicuousness in aposematic species can be advantageous for several reasons. Some studies have shown that high levels of conspicuousness can potentially lead to aposematic prey experiencing higher attack rates than cryptic prey because conspicuousness increases prey visibility to predators, especially when the prey community is exposed to a large number of naïve predators [START_REF] Ruxton | Identifying the ecological conditions that select for intermediate levels of aposematic signalling[END_REF][START_REF] Riipi | Multiple benefits of gregariousness cover detectability costs in aposematic aggregations[END_REF][START_REF] Mappes | Seasonal changes in predator community switch the direction of selection for prey defences[END_REF]. In addition to naïve predators, educated predators may also, at times, exert significant selection pressures on aposematic prey [START_REF] Barnett | State-dependent decision making: educated predators strategically trade off the costs and benefits of consuming aposematic prey[END_REF]. This is because chemically-defended prey contain not only toxins, but also nutrients, and such prey may be profitable to attack, for example when alternative prey are scarce [START_REF] Kokko | Alternative prey can change model-mimic dynamics between parasitism and mutualism[END_REF][START_REF] Lindström | The effect of alternative prey on the dynamic of imperfect Batesian and Müllerian mimicries[END_REF], or when predators do not have a high toxic load [START_REF] Skelhorn | Predators' toxin burders inflluence their strategic decisions to eat toxic prey[END_REF][START_REF] Rowland | Mimicry between unequally defended prey can be parasitic: evidence for quasi-Batesian mimicry[END_REF].

Furthermore, although clearwing butterflies may not be very conspicuous, they are also not entirely cryptic [see 44] as they possess bold and colorful markings on their wings. This is unlikely to be disruptive coloration, as the pattern elements appear to enhance the outline of the wings, and pattern edges have been shown to improve predator avoidance learning [START_REF] Green | Pattern edges improve predator learning of aposematic signals[END_REF]. Moreover, Ithomiini engage in mimetic interactions with other unpalatable butterfly species. Because Müllerian mimicry is the result of convergent selection on a warning display, this suggests that the color pattern in this system is both recognized and avoided by predators. The coloration of transparent Ithomiini butterflies, or lack thereof, could therefore have a cryptic function from a distance and prevent the individual from being seen by predators, but also have an aposematic function once the individual has been seen [START_REF] Barnett | Distance-dependent defensive coloration in the poison frog Dendrobates tinctorius, Dendrobatidae[END_REF][START_REF] Tullberg | Aposematism and crypsis combined as a result of distance dependence: functional versatility of the colour pattern in the swallowtail butterfly larva[END_REF][START_REF] Kang | Body size affects the evolution of hidden colour signals in moths[END_REF]. It is also possible that maximum conspicuousness is not important as long as the signal is distinguishable from the undefended and edible prey, which are typically cryptic, and/or that the signal contrasts with the background [START_REF] Sherratt | The evolution of warning signals as reliable indicators of prey defense[END_REF][START_REF] Ham | Does colour matter? The importance of colour in avoidance learning, memorability and generalisation[END_REF][START_REF] Gamberale-Stille | Contrast versus colour in aposematic signals[END_REF][START_REF] Merilaita | Aposematic signals and the relationship between conspicuousness and distinctiveness[END_REF]. This explanation has been suggested for the zigzag pattern of European vipers (Vipera), which is not very conspicuous, but appears to be a compromise between crypsis and aposematism [START_REF] Niskanen | Significance of the dorsal zigzag pattern of Vipera latastei gaditana against avian predators[END_REF]. As such, a less detectable signal, although potentially less efficient in educating predators, could be beneficial due to its lower detectability, particularly in a situation where predators may kill defended prey.

Chemical defenses in most Ithominii butterflies are believed to be the result of pyrrolizidine alkaloids (PAs) acquired solely pharmacophagously as adults [START_REF] Brown | Adult-obtained pyrrolizidine alkaloids defend ithomiine butterflies against a spider predator[END_REF], and this would certainly explain the large variation in PA content observed in this study. However, our study suggests that PAs may not be the only chemicals responsible for unpalatability in clearwing butterflies. For example, no PAs were found in Methona confusa, despite being strongly unpalatable, and some species may therefore also sequester other chemical compounds from their Solanaceae or Gesneriaceae hostplants. In support of this, larvae of another Methona species (M. themisto) were found to elicit aversive reaction in chicks, suggesting that they contain chemical defences that are potentially sequestered from their hostplants [START_REF] Massuda | Chemical defence of the warningly coloured caterpillars of Methona themisto (Lepidoptera: Nymphalidae: Ithomiinae)[END_REF]. In those species that sequester other toxic compounds, collecting PAs as adults may provide additional protection. Similarly, Ceratinia tutia was the least unpalatable species despite PA concentrations similar to those of other more unpalatable species, suggesting that PA sources may differ in their ability to cause aversion in predators. Whatever the reason, large variations in PA contents such as those observed in our study suggest that individuals within the population may differ in their level of chemical defenses and that in some species younger butterflies may be more palatable as they would not have accumulated sufficient toxins. This may have important repercussions on predator-prey dynamics as the presence of potentially more palatable individuals (i.e. automimics) has been shown to increase sampling by predators [START_REF] Gamberale-Stille | Automimicry destabilizes aposematism: predator sample-and-reject behaviour may provide a solution[END_REF][START_REF] Hansen | Predators use environmental cues to discriminate between prey[END_REF].

In conclusion, we found that transparent Ithomiini butterflies, or clearwing butterflies, are less detectable than their brightly colored counterparts. Although less conspicuous, transparent clearwing butterflies are not any less unpalatable, and may in fact be more unpalatable than opaque species. It seems likely that the color pattern of transparent Ithomiini butterflies is sufficiently distinguishable from palatable prey to trigger recognition and avoidance learning by predators.

The decrease in conspicuousness of transparent wing patterns thus allows butterflies to benefit from both limited detection by predators (crypsis), but also predator deterrence when detected (aposematism). This may be the result of a shift in microhabitat [START_REF] Papageorgis | Mimicry in neotropical butterflies[END_REF], which may alter signal properties or result in differences in the predators that are present or their propensity to attack defended prey. The fact that the transparent species tested were all highly unpalatable suggests that strong unpalatability may decrease selection for conspicuousness and favor the evolution of some forms of crypsis under certain conditions. Strength of unpalatability and potentially some aspects of taste appear to improve memorability in the advent of reduced conspicuousness.

Whether this is also true for the other clades of aposematic butterflies with transparent species that engage in mimicry with Ithomiini species (e.g. Pericopina and Dioptini) is unknown. Further studies focusing on the selective pressures acting on aposematic signaling will improve our understanding of the factors that regulate the interaction between conspicuousness and unpalatability. research permits. We also thank Mario Tuanama, Ronald Mori-Pezo and Willy Daney de Marcillac for their precious help in the field and in the lab. This study was approved by the ethical committee for Animal Welfare of the National Museum of Natural History (ethical declaration n°68-064). Table 1. Average transparency, detectability by avian predators (chromatic contrast in just noticeable difference units [JNDs] for UVS-vision in large gap light conditions), unpalatability, shown as the speed of aversion learning (T) and number of attacks by educated predators (i.e. residual attacks A), both of which increase with increasing palatability, and PA concentration for the 10 taxa of Ithomiini butterflies that were used. The transparent group consist of species with transparency index >20% (i.e. the six last species). The colorful but semi-transparent species Ceratinia tutia was classified as opaque because measures of transmittance were low in any given transparent areas (i.e. although the overall transparency index is relatively high as a result of the surface area of the wing that is transparent, the individual wing areas that are transparent are effectively almost opaque; refer to figure S1) individual per species was used in subsequent analyses, except for those species for which two individuals were measured and for which we used the mean of both individuals.

For the discriminability model, we modelled both vision systems found in birds, i.e. the UVS vision and the VS vision. For UVS vision, we used the spectral data from the blue tit (Cyanistes caeruleus) and relative cone densities of 1:1.9:2.7:2.7 for UVS:S:M:L [2]. For VS vision, we used the spectral data from the shearwater (Puffinus pacificus) [3] with relative cone densities of 1:0.7:1:1.4 for VS:S:M:L [4]. For both vision types, Weber fraction was assumed to be 0.1 for chromatic vision [5,6] and 0.2 for achromatic vision (average of the two species studied in [7]). The blue tit and shearwater were used to model UVS and VS vision type respectively, as they possess a peak in the very short wavelength range similar to that of most other species (i.e.

they are good candidate representatives of both these vision types). Moreover, they are amongst the very few species for which other parameters needed to accurately model bird vision, such as relative cone densities, oil droplet transmission spectra and ocular media transmission spectra, are known [2,3,8].

Transparent areas reflect more in specular configurations than in diffuse configurations (the collection angle is different from the incidence angle), while opaque pigmented areas appear matt, i.e. reflect similar amounts of light in all directions (diffuse). With the measurement configuration and contrast calculations that were used, we likely overestimate the visual contrast between transparent butterflies and their background, making them more similar to opaque butterflies than what likely occurs in nature. Hence, our measurement methods and calculations of contrast are conservative with respect to the potential trend we are testing (i.e. differences in coloration between transparent and opaque butterflies).

bottom for easy cleaning, so as to habituate them to it. Each training period was preceded by a maximum of 2 h of food deprivation. Initially, chicks were placed either in pairs or small groups and allowed to forage from piles of both green and orange palatable pellets presented in a transparent tray with shallow wells. The number of pellets presented in the wells was gradually reduced and at the end of training and during the experiments a plastic tray with 40 pellets was presented. Half of the pellets (i.e. 20 pellets) were of each color, and pellets were placed singly on the tray in alternating colors. All chicks ate readily in the arena at the end of the training and all birds were familiar with the experimental set-up.

Experimental birds were always paired with a "buddy" in the adjacent cage partition so as to minimize stress. Birds had visual, acoustic and even some physical contact through the mesh wire. These buddy chicks did not have access to food during the experiments, so as to avoid distracting the experimental chick, but they were not food deprived prior to use. Also, chicks that were used as buddies were not later used for experiments so as to avoid having chicks with a learned bias. Both experimental birds and their buddies always had ad libitum access to water throughout training and experiments.

Chicks were first tested with all palatable pellets of both colors, and only those that showed no bias were used. Chicks were then presented with a plastic tray with 40 pellets; 20 pellets were of one color and unpalatable and the other 20 pellets were of the other color and palatable. Pellets were placed singly and the colors were alternated so as to avoid chicks attacking pellets of all the same color as a result of being grouped together. Chicks were allowed to attack (peck or eat) 20 pellets before the food tray was removed. This means that in later trials, chicks that had properly learned to avoid unpalatable pellets could eat all 20 palatable pellets and completely avoid the unpalatable ones. Chicks were tested for a total of 12 successive trials, always with the same color combination. In each session we recorded the number of palatable and unpalatable pellets attacked.

By reversing the color association for half the birds that were tested (i.e. for three of the chicks tested with a given butterfly species, we used green experimental pellets, whereas we used orange experimental pellets for the other three chicks, for a total of six chicks tested for each butterfly species), we ensured that there was no color bias (i.e. that the strength of avoidance was not the result of an inherent preference for a given color). At the end of the experiments all chicks were donated to free-range homes.

To assess whether the response of chicks to a given butterfly species differed between individuals, we used the R package rptR to compare the total number of experimental pellets that had been attacked by the six chicks by the end of all 12 trials. Our analysis found them to be significantly similar (Repeatability=44.9%, p<0.001), suggesting that different chicks found a given butterfly species similarly unpalatable. Given that individual chicks varied little in how they reacted to a given butterfly species and that this number was sufficient to be statistically valid, and as per the regulations that govern the ethical use of animals in research, which states that only the minimum number of animals should be used (see: https://www.animalethics.org.au/threers/reduction for reference), no additional chicks were used to test each butterfly species.

For the analysis of whether transparent species as a group differed from opaque species as a group, the colorful but semi-transparent species C. tutia was classified as opaque because its transparency index is lower than that of transparent species (albeit higher than that of opaque species), perception by predators as calculated by our models is more similar to those of opaque species (i.e. although the overall transparency index is relatively high as a result of the surface area of the wing that is transparent, the individual wing areas that are transparent are effectively almost opaque; refer to figure S1 and Table 1), and it belongs to a mimicry ring formed by typically opaque species.

  were collected in north-eastern Peru (San Martín department) in 2015-2017. Collection sites included forest habitats surrounding Tarapoto (Rio Shilcayo basin: 6°27'30"S 76°21'00"W alt 460 m & the Tunel ridge: 6° 27'11"S 76°17'11"W alt 1090 m), Moyobamba (6° 04'34"S 76°57'27"W alt 1130 m), and on Pongo-Baranquita road (6°17'53"S 76°14'38"W alt 200 m).

Figure 2 .Figure 3 .

 23 Figure 2. Relationship between detectability of Ithomiini butterflies by birds (chromatic contrast in just noticeable difference units [JNDs] for UVS-vision), in forest shade (white circles) and large gaps (black circles), and butterfly transparency as measured by the transparency index. Lines were fitted by the method of least squares linear regression for forest shade (dash line: y=-0.028x+2.170; R 2 =0.607) and large gap (solid line: y=-0.097x+7.378; R 2 =0.616). Results of the phylogenetic regressions are shown in Table2.

FiguresFigure S1 .

 S1 Figures

Figure S4 .Figure S5 .

 S4S5 Figure S4. Quantification (μg/mg) of PA content for females (white) and males (grey) of the 10 Ithomiini taxa tested. Species are in order of least transparent (on the left) to most transparent (on the right).

Table 2 .

 2 Butterfly average transparency in relation to average detectability by bird predators for 590 both UVS and VS vision, in both forest shade and large gaps, and for chromatic and achromatic 591 contrasts. Relationships were assessed by performing phylogenetic regressions of detectability on 592 transparency index, accounting for the phylogenetic signal of the residuals (λ).

			Average	Speed of		PA
		Transparency	chromatic	aversion	Residual	concentration
	Species	index (%)	contrast (JNDs)	learning (T)	attacks (A)	(μg/mg)
	Hypothyris ninonia	0.29	7.03	2.47	3.14	23.55
	Tithorea harmonia	0.37	6.53	1.70	0.00	2.83
	Mechanitis polymnia	1.30	6.65	1.61	4.77	23.98
	Ceratinia tutia	17.57	6.37	100.00	10.71	20.83
	Napeogenes inachia	21.10	4.70	0.88	1.11	25.13
	Methona confusa	22.22	3.49	1.03	0.25	0.32
	Godyris zavaleta	24.06	5.07	2.72	0.10	17.76
	Oleria onega	29.35	4.27	0.97	0.36	27.23
	Ithomia salapia aquinia	32.28	4.54	7.54	0.00	23.22
	Ithomia agnosia	38.19	3.90	0.68	0.99	21.69

Table 3 .

 3 Speed of aversion learning (T) in relation to butterfly detectability by bird predators for 596 both UVS and VS vision, in both forest shade and large gaps, and for chromatic and achromatic 597 contrast. Relationships were assessed by performing phylogenetic regressions of T on detectability, 598 accounting for the phylogenetic signal of the residuals (λ).

	599
	Bird	Light	chromatic/		model p-	adjusted			intercept	coefficient
	vision	environment	achromatic	λ	value	R 2	F-stat	df	estimate	estimate
	UVS	forest shade	chromatic	<0.001	0.38	-0.02	0.86	8	-28.94	26.15
	UVS	forest shade achromatic	<0.001	0.92	-0.12	0.01	8	6.31	1.10
	VS	forest shade	chromatic	<0.001	0.46	-0.05	0.60	8	-21.16	23.42
	VS	forest shade achromatic	<0.001	0.92	-0.12	0.009	8	6.49	1.07
	UVS	large gap	chromatic	<0.001	0.28	0.04	1.33	8	-48.89	11.34
	UVS	large gap	achromatic	<0.001	0.97	-0.12	0.002	8	17.59	-0.62
	VS	large gap	chromatic	<0.001	0.43	-0.04	0.68	8	-31.45	8.32
	VS	large gap	achromatic	<0.001	0.96	-0.12	0.0039	8	19.05	-0.78
	600
	601

Table 4 .

 4 Number of attacks by educated predators (A) in relation to detectability for both UVS and VS vision, in both forest shade and large gaps, and for chromatic and achromatic contrast.Relationships were assessed by performing phylogenetic regressions of A on detectability, accounting for the phylogenetic signal of the residuals (λ).

	Bird	Light	chromatic/		model p-	adjusted			intercept	coefficient
	vision	environment	achromatic	λ	value	R 2	F-stat	df	estimate	estimate
	UVS	forest shade	chromatic	<0.001	0.10	0.21	3.46	8	-5.76	5.06
	UVS	forest shade achromatic	<0.001	0.41	-0.03	0.75	8	-2.96	0.10
	VS	forest shade	chromatic	<0.001	0.15	0.15	2.54	8	-4.62	4.78
	VS	forest shade achromatic	<0.001	0.41	-0.03	0.74	8	-2.94	0.10
	UVS	large gap	chromatic	<0.001	0.08	0.25	3.98	8	-8.03	1.90
	UVS	large gap	achromatic	<0.001	0.87	-0.12	0.03	8	-0.29	0.27
	VS	large gap	chromatic	<0.001	0.15	0.14	2.51	8	-6.16	1.59
	VS	large gap	achromatic	<0.001	0.88	-0.12	0.02	8	-0.04	0.24

Table S3 .

 S3 Butterfly average PA content of -a) amount free bases and b) N-oxides, per unit of 779 butterfly weight -in relation to average detectability by bird predators for both UVS and VS vision, 780 in both forest shade and large gaps, and for chromatic and achromatic contrasts. Relationships 781 were assessed by performing phylogenetic regressions of PA content on detectability, accounting 782 for the phylogenetic signal of the residuals (λ).

	783
	784	a)
	Bird	Light	chromatic/		model	adjusted			intercept	coefficient
	vision	environment	achromatic	λ	p-value	R 2	F-stat	df	estimate	estimate
	UVS	forest shade	chromatic	1	0.62	-0.09	0.26	8	8.93	1.39
	UVS	forest shade	achromatic	1	0.73	-0.11	0.12	8	12.10	2.58
	VS		forest shade	chromatic	1	0.62	-0.09	0.26	8	8.91	1.41
	VS		forest shade	achromatic	1	0.82	-0.12	0.05	8	13.63	1.79
	UVS	large gap	chromatic	1	0.59	-0.08	0.31	8	8.47	0.88
	UVS	large gap	achromatic	1	0.72	-0.11	0.14	8	11.98	0.79
	VS		large gap	chromatic	1	0.59	-0.08	0.31	8	8.44	0.89
	VS		large gap	achromatic	1	0.81	-0.12	0.06	8	13.57	0.51
	785
	786	b)
	Bird	Light	chromatic/		model	adjusted			intercept	coefficient
	vision	environment	achromatic	λ	p-value	R 2	F-stat	df	estimate	estimate
	UVS	forest shade	chromatic	1	0.55	-0.07	0.40	8	7.11	1.67
	UVS	forest shade	achromatic	1	0.69	-0.10	0.17	8	11.15	2.94
	VS	forest shade	chromatic	1	0.54	-0.07	0.40	8	7.09	1.68
	VS	forest shade	achromatic	1	0.78	-0.11	0.08	8	12.63	2.23
	UVS	large gap	chromatic	1	0.52	-0.06	0.46	8	6.69	1.04
	UVS	large gap	achromatic	1	0.67	-0.10	0.19	8	11.02	0.90
	VS	large gap	chromatic	1	0.52	-0.06	0.46	8	6.66	1.05
	VS	large gap	achromatic	1	0.77	-0.11	0.09	8	12.56	0.64
	787
	788

Acknowledgements

We thank the Peruvian authorities, and in particular SERFOR (permit: 002-2015-SERFOR-DGGSPFFS & 373-2017-SERFOR-DGGSPFFS), the Gobierno Regional San Martin PEHCBM (permit: 124-2016-GRSM/PEHCBM-DMA/EII-ANP/JARR), the Museo de Historia Natural and Prof. Gerardo Lamas for allowing us to carry out this project and for providing the necessary

Funding. This study was funded by a grant from the Human Frontier Science Program (RGP0014/2016), an ATM grant from the MNHN (TRANSPATOX) and a grant from the French National Research Agency (ANR CLEARWING ANR-16-CE02-0012). MM's postdoc grant was supplied by ANR SPECREP (ANR-14-CE02-0011).

Data

accessibility. Data available from the Dryad Digital Repository:

https://datadryad.org/review?doi=doi:10.5061/dryad.d2h629q [START_REF] Mcclure | Data from: Why has transparency evolved in aposematic butterflies? Insights from the largest radiation of aposematic butterflies, the Ithomiini[END_REF] Transparent species are shown as white circles and conspicuous/non-transparent species are shown as dark circles.

Author contributions

ME designed the study. MM designed the behavioral experiments with the chicks, and MM, CC, MC, LBH and JB performed the experiments in the field and analyzed the data. MM, ME and DG collected specimens for optical and chemical measurements. CD, DG, and CP performed optical measurements and analyses, under the supervision of CA, SB and ME. AM performed chemical measurements, under the supervision of BN, ME and VL. CH performed color pattern imaging and analyses. MM, DG and ME wrote the paper with contributions from all authors.

Competing interests. We have no competing interests.

List of Figures

Supplementary Information

Methods

Optical measurements, transparency and detectability

All measurements of transmittance and reflectance were done using a spectrometer (Starline Avaspec-2048 L, Avantes), fiber optics (FC-UV200-2-1.5 x 40, Avantes) and a deuterium halogen lamp (Avalight DHS, Avantes) emitting in the 300-700 nm range, which covers the entire spectrum visible to birds, assumed to be the main predators. For transmittance, fibers were separated and aligned, and measurements were taken relative to a 'white' reference (no sample) and a 'dark' reference (light source turned off). For reflectance, fibers were gathered into one probe, and measurements were taken perpendicular to the sample, i.e. in a specular configuration, relative to a white reference (Spectralon WS2, Avantes) and a dark reference (light source turned off). All measurements were taken 2mm from the sample.

For 18 of the transparent species, measures of transmittance were taken for two individuals of each species so as to assess repeatability. Transmittance, which is a measure of the average amount of transparency (achromatic component), was computed as the average proportion of light transmitted over the 300-700 nm range and was found to be repeatable within species (R=91.1%, p<0.0001). Moreover, the chroma, which is a measure of the shape (chromatic component) of the spectrum and calculated as the difference between the minimal and maximal transmittance divided by the average transmittance, was also found to be repeatable within species (R=75.1%, p<0.0001).

Repeatability of common chromatic and achromatic descriptors (B2 and S8 in [1]) justifies the use of a single individual as intraspecific variation is minimal and less than that observed between transparent species, and, a fortiori, between transparent and opaque species. As such, one

Experiments measuring unpalatability using domestic chicks

Domestic chicks (Gallus domesticus) used as model predators were obtained from a commercial hatchery and were housed in mixed sex groups of ca. 14 individuals in an outdoor metal cage measuring 2 x 2 x 1.5 m in Tarapoto (San Martín, Peru). As birds were kept outdoors, the ground consisted of natural dirt and wood chips, they had access to natural sunlight from 6AM to 6PM and they were kept at ambient temperatures in the shade. Water and naturally brown chick starter feed (Purina Avemicyn-A®) were provided ad libitum, except during training and experiments.

Pellets of chick feed colored either orange or green were used as prey. Experimental pellets were made by mixing 2.5 g of starter chick feed, ca. 1 g of food coloring or until desired colour was obtained (powder mix, Industria Lucerico SAC), 100 ml of tap water, and 1 g of freshly killed butterflies (equal to ca. 7-20 individuals, depending on the species, of equal sex ratio) mixed into the paste. Whole butterflies were mixed directly into the chick feed so as to remove the natural visual cue (i.e. the butterfly's color pattern) and test the chicks' responses to taste only, thereby comparing the level of averseness between butterfly species. Control pellets consisted of 3.5 g of starter chick feed, water and ca. 1 g of food coloring. Mixtures were left to air dry, broken into small pellets (of ca. 1 mm 3 and ca. 20 mg) and then sieved to ensure that they were all of similar size. A total of six chicks were tested for each butterfly species, and the chicks were never reused, such as, for example, to test another butterfly species.

Birds had no prior experience of unpalatable prey and were initially trained to eat the dyed chicken feed in a clear plastic tray placed on the bottom of the outdoor cage. No food deprivation was necessary at that time as they readily ate the colored chick feed. When chicks were ca. 10 days old, training continued for three days in the experimental set-up, which consisted of a metal chicken wire mesh cage partitioned into two sections measuring 45 x 35 x 50 cm each with a plastic each photo shows the dorsal side of the wings against a dark background so as to highlight transparency, and the right hand side shows the ventral side of the wings against a white background so as to highlight colour patterns.