Mónica Arias
email: moarias@gmail.com

Johanna Mappes

Charlotte Desbois

Swanne Gordon

Melanie Mcclure

Marianne Elias

Ossi Nokelainen

Doris Gomez

Transparency reduces predator detection in mimetic clearwing butterflies

Keywords: aposematic, bird, citizen science, crypsis, detectability, experiment, Ithomiini, vision modelling

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

Introduction

Predation is an important selective pressure and a strong evolutionary force shaping prey colouration. Some prey have evolved colours and textures that mimic those of the background, hence rendering them cryptic [START_REF] Endler | Frequency-dependent predation, crypsis and aposematic coloration[END_REF] and reducing predator detection. In midwater environments, where there is nowhere to hide, crypsis can be achieved by different means, including transparency [START_REF] Johnsen | Hide and seek in the open sea: pelagic camouflage and visual countermeasures[END_REF]. Transparency is common in aquatic organisms where it has been shown to decrease detectability by visual predators, enabling prey to blend in with their environment [START_REF] Kerfoot | A question of taste: crypsis and warning coloration in freshwater zooplankton communities[END_REF][START_REF] Langsdale | Developmental changes in the opacity of larval herring, Clupea harengus, and their implications for vulnerability to predation[END_REF][START_REF] Tsuda | Effect of gut content on the vulnerability of copepods to visual predation[END_REF][START_REF] Zaret | Predators, invisible prey, and the nature of polymorphism in the cladocera (class Crustacea)[END_REF]. By contrast, transparency is generally rare in terrestrial organisms, except for insect wings, which are made of chitin, a transparent material.

However, Lepidoptera (named after ancient Greek words for scalelepisand wingpteron) are an exception as their wings are generally covered with colourful scales that are involved in intraspecific communication [START_REF] Jiggins | Mimicry and the evolution of premating isolation in Heliconius melpomene Linnaeus[END_REF], thermoregulation [START_REF] Miaoulis | Butterfly thin films serve as solar collectors[END_REF], water repellence [START_REF] Wanasekara | Role of surface roughness on wettability and coefficient of restitution in butterfly wings[END_REF], flight enhancement [START_REF] Davis | The redder the better: wing color predicts flight performance in monarch butterflies[END_REF], and antipredator adaptations such as crypsis [START_REF] Stevens | Disruptive coloration, crypsis and edge detection in early visual processing[END_REF], masquerade [START_REF] Suzuki | Gradual and contingent evolutionary emergence of leaf mimicry in butterfly wing patterns[END_REF] and aposematism (i.e. advertisement of unpalatability by the means of conspicuous colouration, [START_REF] Mallet | Individual selection, kin selection, and the shifting balance in the evolution of warning colours: the evidence from butterflies[END_REF].

Ithomiini (Nymphalidae: Danainae), also known as clearwing butterflies, are some of the most abundant butterflies in Neotropical forests [START_REF] Willmott | Maintaining mimicry diversity: optimal warning colour patterns differ among microhabitats in Amazonian clearwing butterflies[END_REF].

Ithomiini species are considered to be unpalatable to some extent due to the accumulation of pyrrolizidine alkaloids collected from Asteraceae, Boraginaceae and Apocynaceae plants [START_REF] Brown | Chemical ecology of dehydropyrrolizidine alkaloids in adult Ithominae(Lepidoptera: Nymphalidae)[END_REF](Brown, , 1985)). Pyrrolizidine alkaloids, naturally present in Ithomiini butterflies, Oreina beetles, or artificially added to mealworms, have been reported to effectively deter predation by birds [START_REF] Brown | Predation on aposematic ithomiine butterflies by tanagers (Pipraeidea melanonota)[END_REF]. Many Ithomiini represent classic examples of aposematic prey, whereby bright wing colour patternsincluding orange, yellow and blackadvertise their unprofitability to predators [START_REF] Mappes | The complex business of survival by aposematism[END_REF][START_REF] Nokelainen | Trade-off between warning signal efficacy and mating success in the wood tiger moth[END_REF][START_REF] Poulton | The colours of animals: their meaning and use, especially considered in the case of insects[END_REF]. Ithomiini butteflies are also involved in mimicry with other aposematic species such as several Heliconius butterflies [START_REF] Beccaloni | Ecology, natural history and behaviour of the Ithomiinae Butterflies and their mimics in Ecuador[END_REF]. Bright contrasting and aposematic colouration is likely to be the ancestral state in the group, since most species in sister lineages (Tellerveni and Danaini) are opaque and aposematic [START_REF] Freitas | Phylogeny of the Nymphalidae (Lepidoptera)[END_REF]. However, transparency has evolved to some degree in approximately 80% of clearwing butterfly species, even though many retain minor opaque and colourful wing elements [START_REF] Beccaloni | Ecology, natural history and behaviour of the Ithomiinae Butterflies and their mimics in Ecuador[END_REF][START_REF] Elias | Mutualistic interactions drive ecological niche convergence in a diverse butterfly community[END_REF][START_REF] Jiggins | The phylogenetic pattern of speciation and wing pattern change in neotropical Ithomia butterflies (Lepidoptera: Nymphalidae)[END_REF]. Similarly to cicadas and damselflies, transparency in these butterfly wings is sometimes enhanced by anti-reflective nanostructures [START_REF] Siddique | The role of random nanostructures for the omnidirectional anti-reflection properties of the glasswing butterfly[END_REF][START_REF] Watson | Putative functions and functional efficiency of ordered cuticular nanoarrays on insect wings[END_REF][START_REF] Yoshida | Antireflective nanoprotuberance array in the transparent wing of a hawkmoth, Cephonodes hylas[END_REF]. Since transparency is often associated with crypsis, for example in aquatic organisms [START_REF] Johnsen | Hide and seek in the open sea: pelagic camouflage and visual countermeasures[END_REF], transparency in these butterfly may decrease detectability by predators.

To determine if transparency in clearwing butterflies decreases detectability by visual predators, we compared predator detection of four Ithomiini species that differ in the amount of transparency of their wings (Fig 1 ): Hypothyris ninonia (largely opaque and brightly coloured), Ceratinia tutia (brightly coloured and translucent), Ithomia salapia (transparent with a pale yellow tint and an opaque contour) and Brevioleria seba (transparent without colouration other than a white band in the forewing and an opaque contour). Given the proportion of light that is transmitted through the butterfly wing of the different species (Fig S1), we predicted that the opaque species Hypothyris ninonia should be the easiest to detect, followed by the translucent species Ceratinia tutia. Finally, the more transparent butterfly species Ithomia salapia and Brevioleria seba should be the least detectable. However, it is also possible that the coloured opaque elements of the transparent species, such as the white band in B. seba and the opaque contour found in most of these species, enhance detection.

We tested our predictions using two complementary behavioural experiments involving birds and humans, and further supported by a vision modelling approach.

Detectability of butterflies was first tested using wild great tits (Parus major) as model bird predators. Great tits are sensitive to UV wavelengths (UVS vision in [START_REF] Ödeen | Evolution of ultraviolet vision in the largest avian radiation -the passerines[END_REF]. Their vision is similar to that of naturally occurring Ithomiini predators such as the houtouc motmot (Momotus momota, [START_REF] Pinheiro | Why do the ithomiines (Lepidoptera, Nymphalidae) aggregate? Notes on a butterfly pocket in central Brazil[END_REF], the fawn-breasted tanager (Pipraeidea melanonota, [START_REF] Brown | Predation on aposematic ithomiine butterflies by tanagers (Pipraeidea melanonota)[END_REF] or the rufous-tailed tanager (Ramphocelus carbo, [START_REF] Brower | Experimental studies of mimicry: Relative palatability and Müllerian mimicry among Neotropical butterflies of the subfamily Heliconiinae[END_REF]. However, unlike Neotropical insectivorous birds, great tits are naïve to ithomiine butterflies and have not learned to associate their colour patterns to toxicity. As a bird's propensity to attack prey is the result of both prey detection and motivation to attack the prey, we also performed behavioural experiments using human participants, which can be useful in disentangling these two factors. Differences in colour perception between great tits and humans include the presence of a fourth single cone type receptor (instead of three cones in humans) that extend the great tits' sensitivity into the UV light spectrum [START_REF] Hart | The visual ecology of avian photoreceptors[END_REF], and oil droplets that refine colour discrimination in birds (Vorobyev, 2003). However, neither humans or birds are able to detect linear polarization, which excludes the use of polarization cues to detect and discriminate between butterfly species (Foster et al., 2018;Greenwood, Smith, Church, & Partridge, 2003;Melgar, Lind, & Muheim, 2015;Montgomery & Heinemann, 1952). Moreover, humans have been found to be good predictors of insect prey survival in the wild [START_REF] Penney | A comparative analysis of the evolution of imperfect mimicry[END_REF]. Finally, models of predator vision (both for birds and humans) were used to complement behavioural experiments and infer the relative detectability of each butterfly species based on their contrast against the background.

Materials and Methods

Butterflies used for the behavioural experiments

Specimens of the four Ithomiini species used in both experimentswhich, in order of increasing transparency are Hypothyris ninonia, Ceratinia tutia, Ithomia salapia aquina, Brevioleria seba (see Figs 1 andS1)were collected in Peru in 2016 and 2017, along the Yurimaguas -Moyobamba road (-6.45°, -76.30°). Butterflies were kept dry in glassine envelopes until use. In behavioural experiments, a single real hindwing and a single real forewing were assembled into artificial butterflies using glue and a thin copper wire to attach the artificial butterfly to a substrate (see Fig S2 for an example). These artificial butterflies mimicked real Ithomiini butterflies at rest, with wings closed and sitting on plant leaves (a typical posture for resting butterflies).

Behavioural experiments using wild birds

Behavioural experiments took place in August and September 2017 at the Konnevesi Research Station (Finland). Thirty wild-caught great tits (Parus major) were used. Birds were caught using spring-up-traps and mist-nets, individually marked with a leg band and used only once. Each bird was housed individually in an indoor cage (65x65x80 cm) and were fed with seeds and water ad libitum, except during training and experiments. During training, birds were given mealworms attached to butterfly wings (see Training section). Birds were deprived of food for up to 2 hours before the experiment to increase their motivation to hunt.

Training. In indoor cages, birds were taught that all four species of butterflies were similarly palatable by offering them laminated wings of four butterflies (one of each species) with a mealworm attached to the copper wire. Wings were laminated during training only, using transparent thin plastic so as to minimize damage and enabling us to re-use the wings between trials. Butterflies were presented to the birds in the absence of vegetation during training so as to enhance the association between butterfly colour patterns and fully edible prey. When birds had eaten all four prey items (one of each species), a new set was presented.

Training ended when birds had eaten 3 sets of butterflies. No time constraint was imposed for training and most birds completed it in less than 4 hours.

In order to familiarise birds with the experimental set-up, which was novel to them, they were released in the experimental cage by groups of two to four birds for approximately one hour the day before the experiment. Oat flakes, seeds and mealworms were dispersed over leaves and vegetation so as to encourage searching for edible items in locations similar to where butterflies would be placed during the experiment.

Experiments. The experimental set-up consisted of a 10m x 10m cage that had tarpaulin walls and a ceiling of whitish dense net that let in natural sunlight. Butterflies were disposed in a 5

x 5 grid, delimited by poles all around the borders and a rope defining rows and columns (see Fig S3). Five specimens of each species (20 specimens in total) were placed in the grid, one per cell. Before each trial, butterflies were photographed over graph paper, used as a scale to measure butterfly size on Image J (Rueden et al., 2017). Butterflies were pinned on top of meadowsweet leaves (Filipendula ulmaria) that had naturally grown in the outdoor cages.

Butterflies were always put in similar places within the cell and could be easily seen from a nearby pole. Butterfly position was randomized but care was taken in 1) leaving the 5 cells closest to the observer empty as birds tended to avoid this area, 2) avoiding having more than two specimens of the same species in the same row or column, and 3) having two specimens of the same species in neighbouring cells. This ensured that all species were evenly represented along the grid. This random configuration was reshuffled between trials.

For each trial, an observer, hidden to the birds, watched from outside the cage through a small window and took notes of which butterfly species were attacked and in which order. A GoPro camera also recorded the experiments. A butterfly was considered detected only if a bird directly approached to attack it, including when the attack failed. No bird was seen hesitating during an attack once it had initiated it. Experiments took place between 9 am and 5 pm. Before each trial, the radiance of ambient light (coming from the sun and sky) was taken by spectrophotometry in the same location. We computed the total radiance (TR) over the bird's spectral sensitivity, which range from 300-700 nm, to account for the intensity of ambient light associated to each experimental trial in the statistical analyses. Further information on weather conditions (cloudy, sunny, etc) was also recorded. Experiments ended when a bird had eaten half of the available butterflies (ie. 10 butterflies) or after 2 hours, whichever happened first. Wings were occasionally re-used if they had not been damaged.

To control for any positional effect on overall species detection, we computed the probability of a bird being present in a given grid area. To do so, a 10-minute interval of each recorded trial was selected and revised to calculate the proportion of time birds spent on the different poles. The time intervals were possible for all trials as they all lasted at least 10 minutes and were selected either as a result of the birds actively attacking prey or actively exploring the cage during that time, based on notes taken by the observer. These probabilities were later used to divide the grid into four main areas according to bird occupancy: furthest and closest corner to the observer, grid border and grid centre (Fig S4a). Most birds fed willingly on all butterflies located on the borders of the grid. Given that butterfly species distribution was random and reshuffled between trials, the four species were similarly represented throughout the grid (Fig S4b), so no bias was expected. For more details about permits, husbandry conditions, training and experiments, see Supplementary Material.

Behavioural experiments using human participants

Between mid-November and early December 2017, visitors of the Montpellier botanical garden (France) were invited to take part in an experiment where they searched for artificial butterflies. Before each trial, participants were shown pictures of various ithomiine butterfly species, both transparent and opaque, different from those used in the experiments to familiarize them with what they would be searching for. Anonymous personal data was collected from each participant, including gender, age group (A1: <10 years, A2: 11-20 y, A3: 21-30 y, A4: 31-40 y, A5: 41-50 y and A6: >51 years), and vision problems. Each participant attempted the experiment only once.

Experimental set-up. As with the behavioural bird experiments, artificial butterflies (N=10 of each of the four species, for a total of 40 butterflies) consisted of one real forewing and one real hindwing assembled with copper wire and placed on leaves, but without the mealworm used in the bird experiments. These butterflies were set-up along two corridors in a forest-like understory habitat of similar vegetation and light conditions. Butterfly order followed a block randomisation, with five blocks each consisting of eight butterflies (i.e. two of each species; see Fig S5). This ensured that observers were similarly exposed to the four species all throughout the experimental transect. Whether a butterfly was placed on the left or right side of the corridor was also randomised and both order and corridor side were changed daily.

Participants could start the path from either end of the set-up and were given unlimited time to complete the trial. However, they could only move forward on the path. Only one participant was allowed in the path at any given time, and they were accompanied by an observer who recorded which butterflies were found. Trials ended when the participant had completed both corridors.

Statistical analyses.

Experiments using birds and humans were analysed independently. Differences in the total number of butterflies of each species that were attacked by predators (for the sake of simplicity we use 'attacked' hereafter for both birds and humans) were compared by fitting generalised linear mixed effect models (GLMM), with bird/human identity as a random factor. A binomial distribution was used for the response variable (attacked or not). For the experiments using birds, butterfly species, butterfly size, trial duration, age and sex of the bird, time to first attack, first butterfly species attacked, butterfly position on the grid (corner -furthest or closest to the observer-, grid border, grid centre), weather (as a qualitative variable), and total radiance (TR), as well as their interactions, were all included as explanatory variables. For human trials, butterfly species, first species attacked, butterfly position, corridor, left or right side of the path, time of day, gender and age of the participant, duration of the experiment, and their interactions, were all used as explanatory variables. In each case, the best fitting model was selected based on minimization of Akaike's Information Criteria (AIC), assuming that models differing by two units or less were statistically indistinguishable (Anderson, Burnham, & White, 1998). Coefficients and standard errors were computed using a restricted maximum likelihood approach and a Wald z test was used to test for factor significance.

In addition to the total number of butterflies attacked per species, an "inconspicuousness" rank was calculated for each butterfly species, as done in a previous study (Ihalainen, Rowland, Speed, Ruxton, & Mappes, 2012). This ranking takes into consideration both the specimens that were attacked and those that were not for each species. Lower values are assigned to those specimens that were attacked (from 1 to 10, according to the sequence of overall prey discovery), and higher values are given to those specimens that were not attacked (all unnoticed specimens are given a value of 11: the maximum number of butterflies that could be attacked before the experiment ended + 1). For example, if a bird captures two H. ninonia second and fifth in the sequence of captured prey, leaving three specimens unnoticed (out of a total of 5 placed in the cage), this species gets a rank value of 2+5+(3x11)=40 for that trial. This inconspicuousness rank distinguishes species attacked first and in higher numbers (lower values of inconspicuousness) from those attacked last and in lower numbers (higher values of inconspicuousness). We fitted a linear mixed effect model to test for differences in rank for each species, assuming a normal distribution, with rank as the response variable. We fitted independent models for birds and human experiments. For bird experiments, bird individual was considered a random factor, and butterfly species, age and sex of the bird, date, time until first attack, first butterfly species attacked, weather as a qualitative variable, and total radiance (TR) were explanatory variables. For humans, participant identity was a random factor, and butterfly species, first species attacked, time of day, gender and age of the participant, duration of the experiment, and their interactions, were all explanatory variables. Again, the best fitting model was selected using AIC minimization.

GLMMs were fitted using nlme [START_REF] Pinheiro | nlme: Linear and nonlinear mixed effects models[END_REF] and lme4 (Bates, Maechler, Bolker, & Walker, 2015, p. 4) packages for R. Moreover, whether specific species were more frequently detected first by either birds or humans was tested using a χ 2 test.

Additionally for birds, we tested whether butterfly location in the grid could explain differences in the overall species' detection, i.e. whether species more likely to be attacked were more often placed on areas more likely to be visited. To do so, the frequency per species on the four different grid zones was compared using a χ 2 . Finally, we tested whether birds and humans created a "search image" (i.e. improved ability in finding butterflies of a given species after encountering a similar one) by counting the number of butterflies of each species attacked consecutively. Results were compared among butterfly species using a χ 2 test. Additionally, whether finding some species improved a bird's or a human's ability to find others was also tested. For each combination of two species, we calculated how many times a butterfly of species 1 was found after a butterfly of species 2. Differences between combinations of butterfly species found by birds were tested using a χ 2 test. For humans, observed results and the frequency at which each possible pair of species was placed consecutively in the original experimental setup were compared using a χ 2 test. All analyses were performed in R (R Foundation for Statistical Computing, 2014).

Colour measures and vision modelling

Finally, models of predator vision (both for birds and humans) were used to complement behavioural experiments and infer the relative detectability of each butterfly species based on their contrast against the background. First, we measured colour (i.e. reflectance) and transmission properties (i.e. transmittance of transparent wing areas) using spectrophotometry. Vorobyev & Osorio's discriminability model (1998) was then used to calculate the contrast between butterfly and background for birds and humans. Detailed methods for measurements and vision modelling can be found in the electronic supplementary material (additional materials and methods).

Results

Behavioural experiments using wild birds

The model that best explained whether butterflies were attacked or not included only the time required before the first attack and the cage area in which the butterfly was located (Table S1). Butterflies were most likely to be attacked when located in the furthest corners and in the borders than in the rest of the cage (z = 9.13, p < 0.001). By contrast, the inconspicuousness rank of a butterfly species was best explained by a model including butterfly species as an explanatory variable (Table S2). Which species was attacked first closely matched wing transmission properties: H. ninonia, the fully opaque species, followed by the translucent C. tutia, the transparent and yellow-tinted I. salapia and the most transparent species in our study, B.seba (X 2 = 11.07, df = 3, p = 0.011; Table S3). Hypothyris ninonia, which was the most colourful species, was usually the first species attacked (t = -3.15, p = 0.002, Fig 2a;

Tables S2 andS3). Species distribution along the four different grid zones was similar (X 2 = 6.19, df = 9, p = 0.72; Fig. S4b).

Generally, birds did not attack several butterflies of the same species consecutively (Fig S6a).

In the rare instances when they did, no differences between species was found (X 2 = 0.6, df = 3, p = 0.90) suggesting that birds did not form a "search image" for any of the butterfly species. No combination of species attacked consecutively at high frequencies were found either (X 2 = 10.88, df = 11, p = 0.45).

Behavioural experiments using human participants

Younger participants found more butterflies than older ones (number of butterflies: z = -2.34, p = 0.019; Fig S7a). Additionally, participants found more butterflies earlier than later in the afternoon (number of butterflies: z = -2.80, p = 0.005; Fig S7a). Generally, the more time participants spent on the experiment, the more butterflies they found (number of butterflies: z = 5.21, p <0.001), although this was most significant for women (number of butterflies: z = -2.96, p = 0.003), Fig S7b). Participants found more butterflies on the corridor that had slightly larger vegetation cover (number of butterflies: z = 3.14, p = 0.002). Participants also found more butterflies at the end rather than at the start of the experiment (number of butterflies: z = 3.70, p < 0.001, Tables S4), most likely because they became accustomed to the set-up and what they were searching for.

Participants were more likely to find opaque butterflies than transparent ones, following the order H. ninonia (H), C. tutia (C), B. seba (B) and I. salapia (I) (H>C, I, B: number of butterflies: z = 5.73, p < 0.001; inconspicuousness rank: t = -3.96, p < 0.001; C>B: inconspicuousness rank: t = -4.81, p < 0.001; B>I: inconspicuousness rank: t = -1.325, p < 0.001; Tables S4 andS5; Fig 2b). However, the gain in detection with increasing time spent searching was highest for the most transparent species (z = -2.75, p = 0.006, Fig S7c).

Hypothyris ninonia was also the species most frequently found first, followed by C. tutia, B. seba and I. salapia (X 2 = 19.5, df = 3, p < 0.001, Table S3). More butterflies of each species were found when C. tutia was found first (t = -3.96, p < 0.001).

There were also differences in the consecutive order in which butterflies were found.

Participants were more likely to find two consecutive butterflies of the same species when they were colourful (H. ninonia -50 times-and C. tutia -58 times) than when they were transparent (B. seba -32 times-or I. salapia -18 times; X 2 = 29.14, df = 3, p < 0.001).

Brevioleria seba and H. ninonia were found consecutively up to four times in a single trial. Some species were also more likely to be found consecutively after another species. The two most opaque butterflies H. ninonia and C. tutia (found 278 times consecutively), and the two transparent species B. seba and I. salapia (found 186 times consecutively), were found consecutively more frequently than any of the other possible combinations after correcting for the number of butterflies found for each species (X 2 = 170.95, df = 5, p < 0.001). These observed frequencies differed significantly from expected as a result of their physical position along the path (X 2 = 79.12, df = 11, p < 0.001, Fig S6b).

Models of bird and human vision

The achromatic weighted contrast between butterfly colour patches and green-leaf background were similar for both birds and humans (mean achromatic contrast for birds: H=3.81, C= 3.15,I=2.31,B=2.11;for humans: H=5.25,C=4.35,I=3.58,B=3.86;Fig S8). For both observers, H. ninonia (the most colourful species) followed by C. tutia (colourful but translucent species) contrasted the most against the leaves, while the transparent butterflies (I. salapia for humans and B. seba for birds) were the least contrasting. Butterflies seem to be more chromatically detectable by birds than for humans (mean chromatic contrast for humans: H = 0.44, C = 0.37, I = 0.25, B = 0.22). For the chromatic contrast seen by birds, C. tutia, followed by H. ninona were the most contrasting, whereas B. seba and I. salapia were the least contrasting (mean chromatic contrast for birds: H = 2.02, C = 2.05, I = 1.30, B = 1.38). For further details of the experiment results, see the Electronic Supplementary Material.

Discussion

Transparency reduces detectability

As initially predicted based on wing transmittance, and as demonstrated by our behavioural experiments and visual modelling results, transparency decreases butterfly detectability.

Interestingly, detection by human participants was similar to that of naïve birds, as shown in other studies [START_REF] Beatty | The evolution of aggregation in profitable and unprofitable prey[END_REF][START_REF] Sherratt | Hierarchical overshadowing of stimuli and its role in mimicry evolution[END_REF], providing further support for using human participants to measure predator detection. Surprisingly, experimental results from the bird experiments differed slightly from predictions based on the measures of transmittance of transparent patches and results obtained from the vision models. For instance, according to the transmittance and the chromatic contrast measured between butterflies and their background, birds should have detected C. tutia more easily than the two more transparent species. Indeed, semi-transparent objects should be more easily detected than fully transparent objects at short distances and when more light is available [START_REF] Johnsen | Transparency and visibility of gelatinous zooplankton from the northwestern Atlantic and Gulf of Mexico[END_REF], such as conditions present during our experiments. Yet this transparent but brightly coloured species was detected at rates similar to those of the most transparent species, perhaps because transparent butterflies were more easily detected and attacked by birds than we predicted (e. g., if an opaque contour enhances detectability of otherwise transparent prey). Alternatively, the semi-transparent C. tutia could have been less detectable by birds, because it shows less strongly delimited contours than those of the most opaque species H. ninonia. Perhaps this hampered its detection as occurs in disruptively coloured prey [START_REF] Honma | Warning coloration can be disruptive: aposematic marginal wing patterning in the wood tiger moth[END_REF][START_REF] Stevens | Disruptive coloration, crypsis and edge detection in early visual processing[END_REF]. These contradicting results highlight the importance of combining both modelling and behavioural experiments to better understand the evolution of transparency and other prey defences.

Transparency in potentially unpalatable butterflies?

Our results demonstrate that transparency can effectively reduce prey detectability in ithomiine butterflies, where several species have been experimentally demonstrated to be chemically-protected (Brown, 1985;[START_REF] Trigo | Pyrrolizidine alkaloids: different acquisition and use patterns in Apocynaceae and Solanaceae feeding ithomiine butterflies (Lepidoptera: Nymphalidae)[END_REF]. This is surprising as aposematic colour patterns, rather than inconspicuousness, are more common in toxic and unpalatable prey [START_REF] Mappes | The complex business of survival by aposematism[END_REF][START_REF] Poulton | The colours of animals: their meaning and use, especially considered in the case of insects[END_REF][START_REF] Ruxton | Avoiding attack: the evolutionary ecology of crypsis, warning signals and mimicry[END_REF]. In fact, conspicuousness is positively correlated with toxicity or unpalatability in some species and can thus be an honest indicator of prey defences [START_REF] Arenas | Signal honesty and predation risk among a closely related group of aposematic species[END_REF][START_REF] Blount | Warning displays may function as honest signals of toxicity[END_REF][START_REF] Maan | Poison frog colors are honest signals of toxicity, particularly for bird predators[END_REF][START_REF] Prudic | Aposematic coloration, luminance contrast, and the benefits of conspicuousness[END_REF][START_REF] Sherratt | The evolution of warning signals as reliable indicators of prey defense[END_REF]. Moreover, predators learn more quickly to avoid unpalatable prey when colours are more conspicuous [START_REF] Gittleman | Why are distasteful prey not cryptic[END_REF][START_REF] Lindstrom | Can aposematic signals evolve by gradual change[END_REF]. This might suggest that the evolution of transparency in these butterflies is the result of a loss or a reduction in unpalatability. If this is the case, the existence of mimicry rings of transparent clearwing butterflies remains unexplained, as this is usually the result of convergence of warning signals promoted by the positive frequencydependent selection exerted by predators [START_REF] Willmott | Maintaining mimicry diversity: optimal warning colour patterns differ among microhabitats in Amazonian clearwing butterflies[END_REF]. Alternatively, if defences are costly, prey may invest in either visual or chemical defences [START_REF] Darst | A mechanism for diversity in warning signals: conspicuousness versus toxicity in poison frogs[END_REF][START_REF] Speed | How bright and how nasty: explaining diversity in warning signal strength[END_REF][START_REF] Wang | Inversely related aposematic traits: reduced conspicuousness evolves with increased toxicity in a polymorphic poison-dart frog[END_REF], as such options have been shown to afford equivalent avoidance by predators [START_REF] Darst | A mechanism for diversity in warning signals: conspicuousness versus toxicity in poison frogs[END_REF]. In which case, transparency should instead be associated with an increase in unpalatability. This relationship between transparency and chemical defences in clearwing butterflies remains to be explored.

Alternatively, transparency may lower detection and function as a primary defence, with aposematism taking over as a secondary defence if the prey is detected. Indeed, transparent butterflies were not completely cryptic for either birds or humans. In fact, although birds detected the most colourful species first, in total they found a similar number of both colourful and transparent butterflies. Moreover, humans appear to learn to detect and perhaps remember common elements between the more transparent species, which might be the result of a search image. As such, Ithomiini butterflies may be cryptic from afar, but perceived as conspicuous from up close. The combination of crypsis and conspicuousness has also been shown for other defended prey [START_REF] Järvi | The cost of being aposematic. An experimental study of predation on larvae of Papilio machaon by the great tit Parus major[END_REF][START_REF] Sillén-Tullberg | Higher survival of an aposematic than of a cryptic form of a distasteful bug[END_REF]. For example, toxic salamanders of the genus Taricha are generally cryptic, only revealing their warning coloured underbelly when threatened [START_REF] Johnson | The selective advantage of the defensive posture of the newt, Taricha granulosa[END_REF]. In Ithomiini, conspicuous elements such as opaque areas that delineate the edges and contrast with the background likely increase detection, as has been shown for artificial moths [START_REF] Stevens | Disruptive coloration, crypsis and edge detection in early visual processing[END_REF]. Furthermore, pigmentary or structurally produced opaque colours, such as the white band in B. seba, may also enhance butterfly detection. This suggests, as do our results and the occurrence of co-mimics in natural habitats, that these butterflies may reduce the cost of conspicuousness using transparency in addition to maintaining the benefits of detectable warning signals. Further behavioural experiments testing the distance at which Ithomiini butterflies are detected are needed to shed further light on the function of aposematism in less conspicuous prey.

Finally, transparency may have evolved as an additional protection against birds such as adult kingbirds (Tyrannus melancholicus, [START_REF] Pinheiro | Palatability and escaping ability in neotropical butterflies: Tests with wild kingbirds (Tyrannus melancholicus, Tyrannidae)[END_REF] which are able to tolerate their chemical defences. Indeed, both theoretical [START_REF] Endler | Predator mixes and the conspicuousness of aposematic signals[END_REF] and experimental [START_REF] Mappes | Seasonal changes in predator community switch the direction of selection for prey defences[END_REF][START_REF] Valkonen | Variation in predator species abundance can cause variable selection pressure on warning signaling prey[END_REF] studies have shown that weak warning signals (not overtly conspicuous) can evolve and be maintained in communities where predators vary in their probability of attacking defended prey. Larvae of Dryas iulia butterflies, pine sawfly larvae (Neodiprion sertifer for example), and shield bugs (Acanthosomatidae, Heteroptera) are only a few of the examples that exist of unpalatable species that display weak visual warning signals (see [START_REF] Endler | Predator mixes and the conspicuousness of aposematic signals[END_REF]. As in the polymorphic poison frog Oophaga granulifera, clearwing species may reflect a continuum between aposematism and crypsis, possibly shaped by differences in the strength of predator selection as a result of the frequency of naïve predators and/or the variation in predator sensitivities to chemical compounds [START_REF] Willink | Not everything is black and white: color and behavioral variation reveal a continuum between cryptic and aposematic strategies in a polymorphic poison frog[END_REF]. A thorough characterization of unpalatability, microhabitat and predator communities would be useful in better understanding conditions that promote the evolution of transparency.

Conclusions

Our study, which combines behavioural experiments with different predators and vision modelling, provides important insights into the complex role transparency may play in predator defences of terrestrial aposematic organisms. We show for the first time that transparency results in the reduction of detectability of terrestrial prey. We also demonstrate that Ithomiini butterflies may in fact be decreasing the costs of conspicuousness, while still retaining visual elements that are recognised as warning signals. Future studies exploring the efficiency of combining transparency and warning signals in decreasing predation risk will further contribute to our understanding of the evolution of cryptic elements in aposematic prey. In order to familiarise birds with the experimental set-up, which was novel to them, they were released in the experimental cage by groups of two to four birds for approximately one hour the day before the experiment. Oat flakes, seeds and mealworms were dispersed over leaves and vegetation to encourage searching for edible items in locations similar to where butterflies would be placed during the experiment. After an hour, no visible oat flakes, seed or mealworms could be found in the cage.

Figures

Experiments. The experimental set-up consisted of a 10m x 10m cage that had tarpaulin walls and a ceiling of whitish dense net that let in natural sunlight. Butterflies were dispersed in a 5

x 5 grid, delimited by poles all around the borders and a rope defining rows and columns (see Fig S3). Two extra poles were placed in the grid centre to increase the appeal of this area for birds. Five specimens of each species (20 specimens in total) were placed in the grid, one per cell. Butterflies were pinned to the top of meadowsweet leaves (Filipendula ulmaria) that had naturally grown in the outdoor cages. Butterflies were always put in similar places within the cell and could be easily seen from a nearby pole. Before the experiment, butterflies were photographed over graph paper, used as a scale to measure butterfly size on Image J (Rueden et al., 2017). Butterfly position was randomized but care was taken in 1) leaving the 5 cells closest to the observer empty as birds tended to avoid this area, 2) avoiding having more than two specimens of the same species in the same row or column, and 3) having two specimens of the same species in neighbouring cells. This ensured that all species were evenly represented along the grid. This random configuration was reshuffled between trials.

For each trial, an observer, hidden to the birds, watched from outside the cage through a small window and took notes of which butterfly species were attacked and in which order. A GoPro camera also recorded the experiments. A butterfly was considered detected only if a bird directly approached to attack it, including when the attack failed. No bird was seen hesitating during an attack once it had initiated it. Experiments took place between 9 am and 5 pm.

Before each trial, the radiance of ambient light (coming from the sun and sky) was measured using an Ocean Optics spectrophotometer in the same location. We computed the total radiance (TR) over the bird's spectral sensitivity, which range from 300-700 nm, to account for the intensity of ambient light associated with each experimental trial in the statistical analyses. Further information on weather conditions (cloudy, sunny, etc) was also recorded.

Experiments ended when a bird had eaten half of the available butterflies (ie. 10 butterflies) or after 2 hours, whichever happened first. Wings were occasionally re-used if they had not been damaged.

To control for any positional effect on overall species detection, we computed the probability of a bird being present in a given grid area. To do so, a 10-minutes interval of each recorded trial was selected and revised to calculate the proportion of time birds spent on the different poles. The time intervals were possible for all trials as they all lasted at least 10 minutes and were selected either as a result of the birds actively attacking prey or actively exploring the cage during that time, based on notes taken by the observer. A total of 87% of all attacks started from the pole closest to the grid cell, while all other attacks were initiated from a pole situated only one grid cell further away. Thus, the probability of visiting a given cell was calculated based on the amount of time spent by the bird on each pole, the number of "close" (immediately next to) or "distant" (one grid cell removed) poles and the probability of visiting them (0.87 for close grid cells and 0.13 for distant grid cells). These probabilities were later used to divide the grid into four main areas according to bird occupancy: furthest and closest corner to the observer, grid border and grid centre (Fig S4a). Most birds fed willingly on all butterflies located on the borders of the grid. Given that butterfly species distribution was random and reshuffled between trials, the four species were similarly represented in those cells (Fig S4b), so no bias was expected.

Colour and optical measurements

Both the transmittance and reflectance of the transparent and opaque wing elements respectively, were measured using spectrophotometry. All measurements were taken using a spectrophotometer (Starline Avaspec-2048 L, Avantes) and a deuterium halogen lamp (Avalight DHS, Avantes) emitting in the 300-700 nm range, including UV, to which birds but not humans are sensitive [START_REF] Chen | Four spectral classes of cone in the retinas of birds[END_REF]. To measure transmittance, illumination and collection fibres were separated (FC-UV200-2-1.5 x 100, Avantes), aligned, and the wing held perpendicularly at an equal distance of ~2mm from each fibre. Measurements were done relative to a white reference (lights turned on with no sample) and a dark reference (light turned off with no sample).

To measure reflectance, an optic probe (FC-UV200-2-1.5 x 100, Avantes) merging illumination and collection angles was used. The fibre was kept in place with a small black chamber that allowed measurements of reflection at 0°, perpendicularly to wing surface.

Samples were again placed at ~2mm from the fibre in front of a light trap to avoid parasitic illumination and reflection. Measurements were relative to a white reference (WS2, Avantes) and a dark reference (light on with the light trap in front). Measurements of both the forewing and the hindwing were taken for one individual of each species. For each wing, 5 measures of transmittance in different transparent areas and 1 measure of reflectance for each colourful patch were taken. Values of transmittance were averaged, and both values of reflectance and transmittance were used to predict butterfly detectability, as a result of the wings' optical properties, by the "predators" used in the two different behavioural experiments.

Models of bird and human vision

We used vision modelling to predict detectability of butterfly species for both birds and humans. Birds and humans are unable to detect linear polarization, and to form spatial images of this property as they do with brightness or colour (Foster et al., 2018;Greenwood, Smith, Church, & Partridge, 2003;Melgar, Lind, & Muheim, 2015;Montgomery & Heinemann, 1952); hence, birds and humans can use only brightness and colour to detect specimens and discriminate between butterfly species.

The contrast perceived by birds and humans for each element of the butterfly colour pattern was calculated using Vorobyev & Osorio's discriminability model (1998). As butterflies were placed on leaves of living plants for all behavioural experiments, they all had green leaves as background. The reflectance of an average green leaf (calculated using the average of 86 different leaves from tropical species [START_REF] Gomez | Simultaneous crypsis and conspicuousness in color patterns: comparative analysis of a neotropical rainforest bird community[END_REF]) transmitted through the transparent wing patches was therefore used. For behavioural experiments using birds as predators, butterflies were seen against leaves in open habitat conditions (under direct sunlight). Hence, for the bird vision model, we used open habitat ambient light conditions (large gaps where sun is visible, similar to conditions present in the outdoor aviaries where we performed bird experiments, [START_REF] Gomez | Simultaneous crypsis and conspicuousness in color patterns: comparative analysis of a neotropical rainforest bird community[END_REF], and blue tit photoreceptors, including oil droplets that enhance colour discrimination [START_REF] Vorobyev | Coloured oil droplets enhance colour discrimination[END_REF], with relative cone densities of 1: 1.92: 2.68: 2.7 for UVS:SWS:MWS:LWS [START_REF] Hart | Visual pigments, oil droplets, ocular media and cone photoreceptor distribution in two species of passerine bird: the blue tit (Parus caeruleus L.) and the blackbird (Turdus merula L.)[END_REF]. We used a Weber fraction of 0.1 for the chromatic response (as reported for Pekin robin Leiothrix lutea in [START_REF] Maier | Colour vision in the passeriform bird, Leiothrix lutea: correlation of visual pigment absorbance and oil droplet transmission with spectral sensitivity[END_REF])) and 0.2 for the brightness response (as the average reported values for known bird species [START_REF] Lind | Brightness discrimination in budgerigars (Melopsittacus undulatus)[END_REF]). In behavioural experiments with humans, we used forest shade ambient light conditions (the forest path we used for the experiment was typical of forest understorey, [START_REF] Gomez | Simultaneous crypsis and conspicuousness in color patterns: comparative analysis of a neotropical rainforest bird community[END_REF], and human photoreceptors (www.cvrl.org ; interpolated every nm) with relative cone densities of 1:16:32 for SWS:MWS:LWS [START_REF] Walraven | A closer look at the tritanopic convergence point[END_REF]. We also used a Weber fraction of 0.018 for LWS in chromatic vision [START_REF] Wyszecki | Color science[END_REF], and 0.11 for brightness [START_REF] Scholtyssek | Brightness discrimination in the harbor seal (Phoca vitulina)[END_REF].

Colour and brightness contrast of butterflies resting on leaves were modelled for both bird and human vision. For transparent wing areas (transparent patches for I. salapia and B. seba), the ambient light was assumed to be transmitted by the wing, reflected on the leaf, and again transmitted by the wing to reach the eye of the observer (see Fig S2). For opaque wing areas (all coloured patches of H. ninonia and C. tutia, and the colourful opaque elements found in the two transparent species), the ambient light had to be reflected by the wing to reach the eye of the observer (see Fig S2). All contrasts were computed using the pavo package [START_REF] Maia | pavo: an R package for the analysis, visualization and organization of spectral data[END_REF] in R (R Foundation for Statistical Computing, 2014). Standardized weighed averages across all areas, weighed for the patch size and standardized for the size of the individuals, were then calculated for chromatic and achromatic contrast between species and green-leaf background.

Detailed statistical analyses and results

Behavioural experiments using wild birds

Birds took anywhere between 1 and 37 minutes (average: 7.54 ± 8.96 min) after release into the experimental cage before initiating an attack. For three of the birds, the experiment ended without having eaten 10 butterflies in the allocated 2 hours. The other 27 birds took between 11 and 112 minutes to attack all 10 butterflies (mean time to attack 10 butterflies: 40.76 ± 26.23 min). For all the trials combined, birds attacked 54% of the H. ninonia butterflies (the most colourful species), 48.7% of the C. tutia (colourful but transparent species), 46.7% of the I. salapia (transparent yellow-tinted butterfly) and 49.3% of the B. seba butterflies (most transparent species).

To test whether birds detected different numbers of butterflies per species, a linear mixed model, including bird ID as a random factor, was fitted. A binomial distribution was used for the response variable (attacked or not), and the butterfly species, butterfly size, trial duration, age and sex of the bird, time to first attack, first butterfly species found, butterfly position on the grid (corner -furthest or closest to the observer-, grid side, grid centre), weather (as a qualitative variable), and total radiance (TR), as well as their interactions, were all selected as explanatory variables. The best fitting model was selected based on minimization of Akaike's Information Criteria (AIC), assuming that models differing by two units or less were statistically indistinguishable (Anderson, Burnham, & White, 1998). The best fitted model, shown in Table S1, included time to first attack, and the position of the butterfly on the grid (furthest or closest corner, border, centre. Fig. S3). According to the results, butterflies were more likely to be attacked when they were in grid zones with a higher probability of a predator being present, when a predator initiated attacks earlier in the experimental trial, and when butterflies were located in the furthest corners from the observer. Thus, similar numbers of butterflies were attacked between species (as species was not part of the best fitting model).

We also calculated an "inconspicuousness rank" that included the order in which butterflies were found and the number of butterflies that were not attacked for each species (i.e. inconspicuousness rank : Ihalainen, Rowland, Speed, Ruxton, & Mappes, 2012). For example, if a bird captured two H. ninonia second and fifth in the sequence of captured prey, this species gets a rank value of 2+5+3x11=40 for that trial. Therefore, highly conspicuous species are characterized by lower inconspicuous rank values. We fitted a linear mixed effect model to test for differences in rank for each species, assuming a normal distribution, with rank as the response variable, bird individual as a random factor and butterfly species, age and sex of the bird, date, time until first attack, first butterfly species found, weather as a qualitative variable, and total radiance (TR) as explanatory variables. Again, the best fitting model was selected using AIC minimization. According to the best fitted generalised linear mixed model, butterfly species explained the variation in inconspicuous rank (Table S2). Butterflies were more conspicuous when they were opaque, such as those belonging to the H. ninonia species.

In addition to the strong spatial distribution effect on butterfly attacks (detected on the number of butterflies found), transparency was found to decrease butterfly detection.

Behavioural experiments using human participants

A total of 102 volunteers participated in the experiment (63 men and 39 women, with 10:11:21:18:31:11 in the A1 (<10): A2 (11-20): A3 (21-30): A4 (31-40): A5 (41-50): A6 (>51) age classes). Of these, 19 volunteers did the experiment before 13h30, 35 between 13h30 and 16h, and 48 after 16h. Participants found between 5 and 28 of the 40 butterflies (12.75 ± 4.68 butterflies found per participant) and took between 7.5 and 37 minutes to complete both corridors (18.04 ±6.5 minutes spent in average per participant). For all the trials combined, participants found 42.5% of the H. ninonia butterflies (the most colourful species), 38% of the C. tutia (colourful but translucent species), 23.54% of the I. salapia (transparent yellow-tinted butterfly) and 28.63% of the B. seba butterflies (most transparent species). Similar statistical analyses were performed for human experiments. First, a linear mixed model was fitted to test for differences in the total number of butterflies per species that were found, assuming a binomial distribution for the response variable (either found or not) and including participant's ID as random factor. Butterfly species, first species found, butterfly position, corridor, left or right side of the path, time of day, gender and age of the participant, duration of the experiment, and their interactions, were all used as explanatory variables. A minimization of Akaike's Information Criteria (AIC) was used to select the best model, assuming that models differing by two units or less were statistically indistinguishable (Anderson et al., 1998). According to the best fitted model (Table S3), participants found more opaque butterflies (H. ninonia) than any other species (z = 5.73, p < 0.001). More butterflies were found earlier than later in the day (z = -2.80, p = 0.005), by men (z = 3.40, p < 0.001) and by younger participants (z = -0.237, p = 0.019). Smaller but significant effects were found for: trial duration, the order in which butterflies were found, and the interactions between species and trial duration, trial duration and gender, and time of day, age and gender.

As in the bird experiments, we also tested whether the order in which butterflies were found, and the number of butterflies that were missed for each species, were related to differences in transparency (i.e. inconspicuousness rank), assuming a Gaussian distribution for the inconspicuousness rank, participant ID as a random factor, and butterfly species, first species found, time of day, gender and age of the participant, duration of the experiment, and their interactions, were all used as explanatory variables. The best fitted linear mixed model (Table S5) shows that the most opaque butterfly species, H. ninonia, was the most conspicuous followed by C. tutia and B. seba. More butterflies were detected when transparent butterflies, especially B. seba and I. salapia, were detected first (t = -12.085, p = 0.004). Fewer butterflies were missed in trials that were done on the second day (t = -1.98, p = 0.05). As for birds, transparency decreases butterfly detection by humans. during the experiment, and a small opening from which birds were released and where the observer could monitor the experiment (its location is indicated as "observer" in the diagram). Dots correspond to poles, which delimited the experimental arena, and rope was used to create the grid layout. Two additional poles were placed in the centre of the arena. A total of 20 artificial butterflies (5 per species) were placed on the green squares (one per square), and never on the "empty" cells, which were avoided by birds, likely due to the proximity of the observer. Cells were divided into four main categories, according to a decreasing probability of being visited by a bird: FC (corner furthest to the observer), grid border, CC (corner closest to the observer) and grid centre. We used a camera, located opposite the observer, to record the experiment.  interactions that affected the total number of butterflies found (see Table S4) and butterfly inconspicuousness rank (see Table S5) were also plotted. As such, regression lines shown in panel a are for the proportion of butterflies found as a function of age for each interval of the time of day (values for these intervals, and p-values testing for slopes different from zero, are:

Figures

<13.5h: r 2 = 0.04, p = 0.206; 13.6h-15.9h: r 2 = 0.314, p = 0.75; >16h: r 2 = -0.013, p = 0.54).

Regression lines shown in panel b are for the proportion of butterflies found as a function of time spent by each gender (Women: r 2 = 0.25, p < 0.001; Men: r 2 = 0.022, p = 0.12).

Regression lines shown in panel c show that time spent on the experiment resulted in higher numbers of butterflies found, especially for the transparent species (H: estimate slope= 0.043, r 2 = 0.014, p = 0.12; C: estimate slope= 0.03, r 2 = 0.005, p = 0.22; I: estimate slope= 0.090, r 2 = 0.12, p < 0.001; B: estimate slope= 0.08, r 2 = 0.07, p = 0.003). Letters in the legend of panel Opaque patches for all species were always considered in reflectance. H. ninonia (H) and C. tutia (C) were modelled under the "reflectance" scenario, while I. salapia (I) and B. seba (B), the transparent species, were modelled under the "transmittance" scenario (see materials and methods).

Figure 1 .Figure 2 .

 12 Figure 1. Dorsal (top row) and ventral (bottom row) view of butterfly species used in the

Figure S1 .Figure S2 .

 S1S2 Figure S1. Average transmittance values per butterfly species: the lower the value, the more

Figure S4 .

 S4 Figure S4. Probability of a bird occupying different grid zones (a) and distribution of butterfly

Figure S5 .

 S5 Figure S5. Example of an experimental trial with human participants. Numbers represent the

Figure S6 .

 S6 Figure S6. Frequency of pairs of butterflies of the same (left side of the dash line) or different

Figure S7 .

 S7 Figure S7. Correlation between the proportion of butterflies found by human participants and

c

  stand for species names: H.ninonia (H), C.tutia (C), I. salapia (I), and B. seba (B). Butterfly transparency increases from top to bottom of the legend (i.e. H<C<I<B).

Figure S8 .

 S8 Figure S8. Chromatic (DS, plots on the left) and achromatic (DQ, plots on the right) contrasts

Table S2 .

 S2 Linear mixed model (LMM) results for the best-fitting model explaining the inconspicuousness rank for each species used in the bird experiments. Butterfly species was the explanatory variable. Species from most opaque to most transparent are H. ninonia (H)> C. tutia (C) > I. salapia (I)~ B. seba (B). Symbols: ** p<0.01, ***p<0.001.

	Explanatory Variables	Estimate	Std Error t value	p
	Intercept	41.775	0.539	77.53	<0.001 ***
	Species.H>C,I,B	-0.981	0.311	-3.15	0.002 **
	Species.C>B	0.411	0.880	0.47	0.641
	Species.I>B	-0.455	0.880	-0.52	0.606

Table S3 .

 S3 The number of times a species was found first in a given trial, either by birds or humans.

	Species	Birds Humans
	H. ninonia 15	43
	C. tutia	7	27
	I. salapia 5	14
	B. seba	3	18

Table S4 .

 S4 GLMM results for the best-fitting model explaining the number of butterflies found by human observers (binomial distribution). Explanatory variables are: corridor, species, time of day, participant age and gender, order of butterfly position in the experimental sequence (butterfly block), time spent on the experiment, and the following interactions: species and time spent on the experiment, time of day, and participant age and gender, and time of day and gender. Butterfly species, from most opaque to most transparent are (H > C > I ~ B or H. ninonia > C. tutia > I. salapia ~ B. seba). z corresponds to the values from the Wald z test used to test for factor significance. Symbols: * p <0.05, ** p<0.01, ***p<0.001.

	Expalnatory variables	Estimate Std. Error z	p
	Intercept	-1.311	0.380	-3.45	<0.001 ***
	Corridor	0.223	0.071	3.14	0.002 **
	Species. H > C, I, B	0.616	0.108	5.73	<0.001 ***
	Species. C > B	0.004	0.141	0.03	0.976
	Species. B > I	0.221	0.162	1.37	0.171
	Time of day	-0.416	0.149	-2.80	0.005 **
	Gender M	1.031	0.304	3.40	<0.001 ***
	Age Group	-0.237	0.101	-2.34	0.019 *
	Time spent (min)	0.056	0.011	5.21	<0.001 ***
	Butterfly block	0.092	0.025	3.70	<0.001 ***
	Species. H > C, I, B: Time spent	-0.015	0.005	-2.75	0.006 **
	Species. C > B: Time spent	0.005	0.007	0.69	0.488
	Species. I > B: Time spent	0.004	0.008	0.56	0.573
	Time spent : Gender M	-0.043	0.014	-2.96	0.003 **
	Time of day: Gender F: Age Group 0.097	0.043	2.27	0.023 *
	Time of day: Gender M: Age Group 0.047	0.039	1.22	0.223

Acknowledgments

We thank Tuuli Salmi and Tiffanie Kortenhoff for their invaluable help with behavioural experiments, Helinä Nisu for her advice on bird care, SERFOR, Proyecto Huallaga and Gerardo Lamas for providing research permits in Peru (collecting and exportation permit 002-2015-SERFOR-DGGSPFFS), as well as Corentin Clerc, Monica Monllor, Alexandre Toporov and Marc Toporov-Elias for help with collecting butterflies used in this study, Céline Houssin for calculations of wing surfaces for each butterfly colour pattern patch and for Ithomiini pictures, Konnevesi Research Station, which provided the facilities used for bird experiments, and visitors to Montpellier botanical garden for their enthusiastic contribution.

We thank Marcio Cardoso and another anonymous reviewer for their helpful comments and suggestions. The study was funded by the Academy of Finland (Grants 2100000256 and 21000038821), the Clearwing ANR program (ANR-16-CE02-0012) and the Human Frontier Science Program grant (RGP 0014/2016).

Author contribution

DG, ME, JM and MA designed the study; ME, MM and DG collected the butterfly samples; MA, SG, ON, ME and JM performed the experiments; DG and CD did the optical measurements; MA, DG and ME analysed the data; MA, DG, MM, ME, ON, SG and JM wrote the manuscript. Authors have none conflict of interest to declare.

Data available from the Dryad Digital Repository [START_REF] Arias | Data from: Transparency reduces predator detection in mimetic clearwing butterflies[END_REF].

Electronic supplementary Material

Additional materials and methods and results.

Detailed behavioural experiments

Behavioural experiments using wild birds

Behavioural experiments took place in August and September 2017 at the Konnevesi Research Station (Finland) under permit from the National Animal Experiment Board (ESAVI/9114/04.10.07/2014) and the Central Finland Regional Environment Centre (VARELY/294/2015). Thirty wild-caught great tits (Parus major) were used, including 3 juveniles and 10 adult females, and 8 juveniles and 9 adult males. Birds were caught using spring-up-traps and mist-nets, individually marked with a leg band and used only once. Each bird was housed individually in an indoor cage (65x65x80 cm), with a 12:12 photoperiod.

Birds were fed with peanuts, sunflower seeds, oat flakes and water ad libitum, except during training and experiments. During training, birds were given mealworms attached to butterfly wings (see Training section). Birds were deprived of food for up to 2 hours before the experiment to increase their motivation to hunt. Most birds were kept in captivity for less than a week, after which they were released at their capture site.

Training. In indoor cages, birds were taught that all four species of butterflies were similarly palatable by offering them laminated wings of four butterflies (one of each species) with a mealworm attached to the copper wire. Wings were laminated during training only, using transparent thin plastic so as to minimize damage and enabling us to re-use the wings between trials. Butterflies were presented to the birds in the absence of vegetation during training so as to enhance the association between butterfly colour patterns and fully edible prey. When birds had eaten all four of the prey items (one of each species), a new set was presented. S3). Comparisons correspond to: 1) more attacks on the grid borders and the corners located furthest from the observer than on the rest of the grid, 2) more attacks in corners than on the grid borders, 3) more attacks on corners located closest to the observer than in the centre of the grid. z corresponds to the values from the Wald z test used to test for factor significance. Symbols: ***p<0.001, *p<0.05.