Transparency reduces predator detection in mimetic clearwing butterflies

Abstract

Predation is an important selective pressure, and some prey have evolved conspicuous warning signals that advertise unpalatability (i.e., aposematism) as an antipredator defence. Conspicuous colour patterns have been shown effective as warning signals, by promoting predator learning and memory. Unexpectedly, some butterfly species from the unpalatable tribe Ithomiini possess transparent wings, a feature rare on land but common in water, known to reduce predator detection.
We tested whether transparency of butterfly wings was associated with decreased detectability by predators, by comparing four butterfly species exhibiting different degrees of transparency, ranging from fully opaque to largely transparent. We tested our prediction using both wild birds and humans in behavioural experiments. Vision modelling predicted butterfly detectability to be similar for these two predator types.
In concordance with predictions, the most transparent species were almost never found first and were detected less often than the opaque species by both birds and humans, suggesting that transparency enhances crypsis. However, humans were able to learn to better detect the more transparent species over time.
Our study demonstrates for the first time that transparency on land likely decreases detectability by visual predators.

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© 2019 The Authors. Functional Ecology
© 2019 British Ecological Society
1 | INTRODUCTION
Predation is an important selective pressure and a strong evolution‐
ary force shaping prey coloration. Some prey have evolved colours
and textures that mimic those of the background, hence rendering
them cryptic (Endler, 1988) and reducing predator detection. In mid‐
water environments, where there is nowhere to hide, crypsis can
be achieved by different means, including transparency (Johnsen,
2014). Transparency is common in aquatic organisms where it has
been shown to decrease detect ability by visual predators, enabling
prey to blend in with their environment (Kerfoot, 1982; Langsdale,
1993; Tsuda, Hiroaki, & Hirose, 1998; Zaret, 1972). 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 scale‐
lepis and wing‐pteron) are an exception as their wings are gener‐
ally covered with colourful scales that are involved in intraspecific
communication (Jiggins, Estrada, & Rodrigues, 2004), thermoreg‐
ulation (Miaoulis & Heilman, 1998), water repellence (Wanasekara
& Chalivendra, 2011), flight enhancement (Davis, Chi, Bradley, &
Received: 5 September 2018
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Accepted: 14 January 2019
DOI : 10.1111 /1365 ‐2435 .13315
RESEARCH ARTICLE
Transparency reduces predator detection in mimetic clearwing
butterflies
Mónica Arias1 | Johanna Mappes2 | Charlotte Desbois3 | Swanne Gordon2 |
Melanie McClure3 | Marianne Elias3* | Ossi Nokelainen2* | Doris Gomez1,4*
*Co‐las t authors.
1Univ Montpellier, Univ Paul Valéry
Montpe llier 3, EPHE, IRD, CEFE, Montp ellier,
France
2Department of Biological and
Environmental Science, Centre of Excellence
in Biolog ical Interact ions, Universit y of
Jyväskylä, Jyväskylä, Finland
3Institut de Systématique, Evolution,
Biodive rsité (IS YEB), CNRS, MNHN,
Sorbonne Université, EPH E, Université des
Antilles, Paris, France
4INSP, Sorbonne Univer sité, CNR S, Pari s,
France
Correspondence
Mónica Arias
Email: moarias@gmail.com
Funding information
Suomen Akatemia, Grant/Award Number:
2100000256 and 21000038821; Agence
Nationale de la Recherche, Grant/Award
Number: ANR-16-CE02-0012; Human
Frontier Science Program, Grant/Award
Number : RGP 0014/2016
Handling Editor: Caroline Isaksson
Abstract
1. Predation is an important selective pressure, and some prey have evolved con‐
spicuous warning signals that advertise unpalatability (i.e., aposematism) as an an‐
tipredator defence. Conspicuous colour patterns have been shown effective as
warning signals, by promoting predator learning and memory. Unexpectedly, some
butterfly species from the unpalatable tribe Ithomiini possess transparent wings, a
feature rare on land but common in water, known to reduce predator detection.
2. We tested whether transparency of butterfly wings was associated with de‐
creased detectability by predators, by comparing four butterfly species exhibiting
different degrees of transparency, ranging from fully opaque to largely transpar‐
ent. We tested our prediction using both wild birds and humans in behavioural
experiments. Vision modelling predicted butterfly detectability to be similar for
these two predator types.
3. In concordance with predictions, the most transparent species were almost never
found first and were detected less often than the opaque species by both birds
and humans, suggesting that transparency enhances crypsis. However, humans
were able to learn to better detect the more transparent species over time.
4. Our study demonstrates for the first time that transparency on land likely de‐
creases detectability by visual predators.
KEYWORDS
aposematic, bird, citizen science, crypsis, detectability, experiment, Ithomiini, vision modelling

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Altizer, 2012), and antipredator adaptations such as crypsis (Stevens
& Cuthill, 2006), masquerade (Suzuki, Tomita, & Sezutsu, 2014) and
aposematism (i.e., advertisement of unpalatability by the means of
conspicuous coloration, Mallet & Singer, 1987).
Ithomiini (Nymphalidae: Danainae), also known as clearwing but‐
terflies, are some of the most abundant butter flies in Neotropic al for‐
ests (Willmott, Willmott, Elias, & Jiggins, 2017). Ithomiini species are
considered to be unpalatable to some extent due to the accumulation
of pyrrolizidine alkaloids collected from Asteraceae, Boraginaceae
and Apocynaceae plants (Brown, 1985). Pyrrolizidine alkaloids, nat‐
urally present in Ithomiini butterflies, Oreina beetles, or artificially
added to mealworms, have been reported to effectively deter preda‐
tion by birds (Brown & Neto, 1976). Many Ithomiini represent classic
examples of aposematic prey, whereby bright wing colour patterns—
including orange, yellow and black—advertise their unprofitability
to predators (Mappes, Marples, & Endler, 2005; Nokelainen, Hegna,
Reudler, Lindstedt, & Mappes, 2012; Poulton, 1890). Ithomiini butter‐
flies are also involved in mimicry with other aposematic species such
as several Heliconius butterflies (Beccaloni, 1997). Bright contrasting
and aposematic coloration is likely to be the ancestral state in the
group, since most species in sister lineages (Tellerveni and Danaini)
are opaque and aposematic (Freitas & Brown, 2004). However, trans‐
parency has evolved to some degree in approximately 80% of clear‐
wing butterfly species, even though many retain minor opaque and
colourful wing elements (Beccaloni, 1997; Elias, Gompert, Jiggins, &
Willmott, 2008; Jiggins, Mallarino, Willmott, & Bermingham, 2006).
Similarly to cicadas and damselflies, transparency in these butter‐
fly wings is sometimes enhanced by anti‐reflective nanostructures
(Siddique, Gomard, & Hölscher, 2015; Watson, Myhra, Cribb, &
Watson, 20 08; Yoshida, Motoyama, Kosaku, & Miyamoto, 1997).
Since transparency is often associated with crypsis, for example in
aquatic organisms (Johnsen, 2014), transparency in these butterflies
may decrease detectability by predators.
To determine whether transparency in clearwing butterflies
decreases detectabilit y by visual predators, we compared preda‐
tor detection of four Ithomiini species that differ in the amount of
transparency of their wings (Figure 1): Hypothyris ninonia (lar gely
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 with‐
out coloration 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 (Supporting
Information Figure S1), we predicted that the opaque species
H. ninonia should be the easiest to detect, followed by the trans‐
lucent species C. tutia. Finally, the more transparent butterfly spe‐
cies I. salapia and B. seba should be the least detectable. However,
it is also possible that the coloured opaque elements of the trans‐
parent 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 ex‐
periments involving birds and humans and further suppor ted by a
vision modelling approach.
Detect ability 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 Ödeen, Håstad, & Alström, 2011). Their
vision is similar to that of naturally occurring Ithomiini predators such
as the houtouc motmot (Momotus momota, Pinheiro, Medri, & Salcedo,
2008), the fawn‐breasted tanager (Pipraeidea melanonota, Brown &
Neto, 1976), or the rufous‐tailed tanager (Ramphocelus carbo, Browe r,
Brower, & Collins, 1963). However, unlike Neotropical insectivorous
birds, great tits are naïve to ithomiine but terflies and have not learned
to associate their colour patterns to toxicity. As a bird’s propensity to
attac k prey is the result of bot h prey detection an d motivation to att ack
the prey, we also performed behavioural experiments using human
participants, which can be useful in disentangling these two factors.
FIGURE 1 Dorsal (top row) and ventral (bottom row) view of butterfly species used in the study (photographed against a black and
a white back ground to show the location and degree of transparency in the wings). Wing transparency (transmission and area occupied
by transparent patches) increases from lef t (most opaque) to right (most transparent): Hypothyris ninonia (largely opaque), Ceratinia tutia
(translucent but brightly coloured), Ithomia salapia (transparent with a pale yellow tint and black wing contour), Brevioleria seba (transparent
without coloration other than a white band in the forewing and a black wing contour). © Céline Houssin

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Differences in colour perception between great tits and humans in‐
clude the presence of a fourth single cone t ype receptor (instead of
three cones in humans) that extend the great tits’ sensitivity into the
UV light spectrum (Hart, 2001) and oil droplets that refine colour dis‐
crimination in birds (Vorobyev, 2003). However, neither humans nor
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; Montgomer y & Heinemann,
1952). Moreover, humans have been found to be good predic tors of
insect prey survival in the wild (Penney, Hassall, Skevington, Abbott, &
Sherratt, 2012). Finally, models of predator vision (both for birds and
humans) were used to complement behavioural experiments and infer
the relative detectabilit y of each butterfly species based on their con‐
trast against the background.
2 | MATERIALS AND METHODS
2.1 | Butterflies used for the behavioural
experiments
Specimens of the four Ithomiini species used in both experiments—
which, in order of increasing transparency, are H. ninonia, C. tutia,
I. salapia aquina, B. seba (see Figure 1 and Supporting Information
Figur e S1)—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 Supporting Information
Figure S2 for an example). These artificial butter flies mimicked real
Ithomiini butter flies at rest, with wings closed and sitting on plant
leaves (a typical posture for resting butterflies).
2.2 | 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
(P. 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 (65 × 65 × 80 cm) and was
fed with seeds and water ad libitum, except during training and ex‐
periments. During training, birds were given mealworms attached to
butter fly wings (see Training section). Birds were deprived of food for
up to 2 hrs before the experiment to increase their motivation to hunt.
2.2.1 | Training
In indoor cages, birds were taught that all four species of butterflies
were similarly palatable by offering them laminated wings of four
butter flies (one of each species) with a mealworm attached to the
copper wire. Wings were laminated during training only, using trans‐
parent thin plastic so as to minimize damage and enabling us to reuse
the wings between trials. Butterflies were presented to the birds in
the absence of vegetation during training so as to enhance the as‐
sociation between butter fly 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 three sets
of butter flies. No time constraint was imposed for training, and most
birds completed it in < 4 hrs.
In order to familiarize 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 1 hr 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 but terflies would be placed during the
experiment.
2.2.2 | Experiments
The experimental set‐up consisted of a 10 m × 10 m cage that had
tarpaulin walls and a ceiling of whitish dense net that let in natu‐
ral sunlight. Butterflies were disposed in a 5 × 5 grid, delimited by
poles all around the borders and a rope defining rows and columns
(see Supporting Information Figure 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 ImageJ (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 c are was taken in (a) leaving the five cells
closest to the obser ver empty as birds tended to avoid this area,
(b) avoiding having more than two specimens of the same species
in the same row or column and (c) having two specimens of the
same species in neighbouring cells. This ensured that all species
were evenly represented along the grid. This random configura‐
tion 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 a.m. and 5 p.m.
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 ranges from 300 to 700 nm, to account for the intensity of
ambient light associated with each experimental trial in the statis‐
tical analyses. Further information on weather conditions (cloudy,
sunny, etc) was also recorded. E xperiments ended when a bird had
eaten half of the available butterflies (i.e., 10 butterflies) or after
2 hrs, whichever happened first. Wings were occasionally reused if
they had not been damaged.

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To control for any positional effect on overall species detec tion,
we computed the probability of a bird being present in a given grid
area. To do so, a 10‐min 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 min 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 probabili‐
ties 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 (Suppor ting Information Figure S4a). Most
birds fed willingly on all butterflies located on the borders of the
grid. Given that butterfly species distribution was random and re‐
shuffled between trials, the four species were similarly represented
throughout the grid (Suppor ting Information Figure S4b), so no bias
was expected. For more details about permits, husbandry condi‐
tions, training and experiments, see Supporting Information.
2.3 | 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 search‐
ing for. Anonymous personal data were collected from each partici‐
pant, including gender, age group (A1: <10 years, A2: 11–20 years, A3:
21–30 years, A4: 31–40 years, A5: 41–50 years and A6: >51 years) and
vision problems. Each participant attempted the experiment only once.
2.3.1 | 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) con‐
sisted 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 understorey habitat of similar vegetation
and light conditions. Butterfly order followed a block randomization,
with five blocks each consisting of eight butterflies (i.e., two of each
species; see Supporting Information Figure 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 randomized and both order
and corridor side were changed daily. Participants could star t 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
butter flies were found. Trials ended when the participant had com‐
pleted both corridors.
2.4 | Statistical analyses
Experiments using birds and humans were analysed independently.
Differences in the total number of butterflie s 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 gener‐
alized linear mixed‐ef fect models (GLMM), with bird/human identity as
a ran dom factor. A binomial distribution was used for the respo nse var‐
iable (att acked or not). For the experiment s using birds, butterfly spe‐
cies, but terfly 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 TR, as well as their interactions,
were all included as explanator y variables. For human trials, butterfly
species, first species at tacked, butterfly position, corridor, left or right
side of the path, time of day, gender and age of the participant, dura‐
tion of the experiment and their interactions were all used as explana‐
tory variables. In each case, the best fitting model was selected based
on minimization of Akaike’s information criteria (AIC), assuming that
models di ffering by two uni ts or less were statistic ally indistinguishable
(Anderson, Burnham, & White, 1998). Coefficients and st anda rd 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 spe‐
cies, 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 discover y), 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 butter flies that could be attacked before
the experiment ended + 1). For example, if a bird captures two H. ni-
nonia second and fifth in the sequence of captured prey, leaving
three specimens unnoticed (out of a total of five placed in the cage),
this species gets a rank value of 2 + 5 + (3 × 11) = 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 inconspicuous‐
ness). 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 butter fly species attacked,
weather as a qualitative variable and TR were explanatory variables.
For humans, participant identity was a random factor, and but terfly
species, first species at tacked, time of day, gender and age of the
participant, duration of the experiment and their interactions were
all explanator y variables. Again, the best fitting model was selected
using AIC minimization. GLMMs were fitted using nlme (Pinheiro,
Bates, DebRoy, Sarkar, & R Core team, 2009) and lme4 (Bates,

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Maechler, Bolker, & Walker, 2014, p. 4) packages for R. Moreover,
whether specific species were more frequently detected first by ei‐
ther birds or humans was tested using a chi‐square test.
Additionally for birds, we tested whether butterfly location in the
grid could explain differences in the overall species’ detection, that is
whether species more likely to be attacked were more of ten 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 chi‐square test .
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 but‐
terflies of each species attacked consecutively. Results were com‐
pared among butterfly species using a chi-square 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
butter fly species found by birds were tested using a chi‐square test.
For humans, observed result s and the frequency at which each pos‐
sible pair of species was placed consecutively in the original exper‐
imental set-up were compared using a chi-square test. All analyses
were performed in R (R Foundation for Statistical Computing, 2014).
2.5 | Colour measures and vision modelling
Finally, models of predator vision (both for birds and humans) were
used to complement behavioural experiments and infer the rela‐
tive detec tability of each butter fly 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 and Osorio’s discrimi‐
nability model (1998) was then used to calculate the contrast be‐
tween butterfly and background for birds and humans. Detailed
methods for measurements and vision modelling can be found in the
Supporting Information (additional materials and methods).
3 | RESULTS
3.1 | 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 (Supporting Information
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
butter fly species was best explained by a model including butterfly
species as an explanatory variable (Supporting Information Table S2).
Which species was attacked first closely matched wing transmis‐
sion 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 (Χ2 = 11.07, df = 3,
p = 0.011; Supporting Information Table S3). Hypothyris ninonia, which
was the most colourful species, was usually the first species attacked
(t = −3.15, p = 0.002, Figure 2a; Supporting Information Tables S2 and
S3). Species distribution along the four different grid zones was similar
(Χ2 = 6.19, df = 9, p = 0.72; Supporting Information Figure S4b).
Generally, birds did not at tack several but terflies of the same
species consecutively (Supporting Information Figure S6a). In the
rare instances when they did, no differences between species were
found (Χ2 = 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 (Χ2 = 10.88, df = 11, p = 0.45).
3.2 | Behavioural experiments using human
participants
Younger part icipants found m ore butterf lies than older on es (number
of butter flies: z = −2.34, p = 0.019; Supporting Information Figure
S7a). Additionally, participants found more butterflies earlier than
FIGURE 2 Sum of the inconspicuousness rank for each butterfly species calculated from the behavioural experiment s using (a)
great tits and (b) humans. Species for which butter flies were detected first and most of ten by birds or humans have lower values of
“inconspicuousness rank.” But terfly transparency increases from left to right: Hypothyris ninonia (H), Ceratinia tutia (C), Ithomia salapia (I) and
Brevioleria seba (B). Letters above the bars mean significant differences below 0.05

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later in the af ternoon (number of butter flies: z = −2 . 80, p = 0.005;
Suppor ting Information Figure S7a). Generally, the more time par‐
ticipant s spent on the experiment, the more but terflies 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,
Supporting Information Figure S7b). Participants found more butter‐
flies on the corridor that had slightly larger vegetation cover (number
of butterflies: z = 3.14, p = 0.002). Participants also found more but‐
terflies at the end rather than at the start of the experiment (number
of butterflies: z = 3.70, p < 0.001, Supporting Information 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 ©,
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: incon‐
spicuousness rank: t = −1.325, p < 0.001; Supporting Information
Tables S4 and S5; Figure 2b). However, the gain in detection with
increasing time spent searching was highest for the most transpar‐
ent species (z = −2.75, p = 0.006, Supporting Information Figure
S7c). Hypothyris ninonia was also the species most frequently
found first, followed by C. tutia, B. seba and I. salapia (Χ2 = 19. 5,
df = 3, p < 0.001, Supporting Information Table S3). More but‐
terflies 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
butter flies were found. Participants were more likely to find two
consecutive butterflies of the same species when they were co‐
lourful (H. ninonia—50 times‐ and C. tutia—58 times) than when
they were transparent (B. seba—32 times‐ or I. salapia—18 t i m e s ;
Χ2 = 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 spe‐
cies. The two most opaque but terflies 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 consecu‐
tively more frequently than any of the other possible combinations
after correcting for the number of butterflies found for each spe‐
cies (Χ2 = 170.95, df = 5, p < 0.001). These observed frequencies
differed significantly from expected as a result of their physical
position along the path (Χ2 = 79.12, df = 11, p < 0.001, Supporting
Information Figure S6b).
3.3 | Models of bird and human vision
The achromatic‐weighted contrast between butter fly colour
patches and green‐leaf background was 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;
Suppor ting Information Figure S8). For both obser vers, H. ninonia
(the most colourful species) followed by C. tutia (colourful but trans‐
lucent species) contrasted the most against the leaves, while the
transparent butterflies (I. salapia for humans and B. seba for birds)
were the least contrasting. Butter flies seem to be more chromati‐
cally 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. ninonia were the most
contrasting, whereas B. seba and I. salapia were the least contrast‐
ing (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
Supporting Information.
4 | DISCUSSION
4.1 | Transparency reduces detectability
As initially predicted based on wing transmittance, and as dem‐
onstrated by our behavioural experiments and visual model‐
ling results, transparency decreases butterfly detectability.
Interestingly, detection by human participants was similar to that
of naïve birds, as shown in other studies (Beatty, Bain, & Sherratt,
2005; Sherratt, Whissell, Webster, & Kikuchi, 2015), providing
further support for using human par ticipants to measure predator
detection. Surprisingly, experimental results from the bird experi‐
ments 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 bet ween butterflies and
their background, birds should have detected C. tutia more easily
than the two more transparent species. Indeed, semi‐transpar‐
ent objects should be more easily detec ted than fully transpar‐
ent objects at short distances and when more light is available
(Johnsen & Widder, 1998), 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 eas‐
ily 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 delim‐
ited contours than those of the most opaque species H. ninonia.
Perhaps this hampered its detection as occurs in disruptively
coloured prey (Honma, Mappes, & Valkonen, 2015; Stevens &
Cuthill, 2006). These contradicting results highlight the impor‐
tance of combining both modelling and behavioural experiments
to better understand the evolution of transparency and other
prey defences.
4.2 | Transparency in potentially unpalatable
butterflies?
Our results demonstrate that transparency can effectively re‐
duce prey detectability in ithomiine butterflies, where several
species have been experimentally demonstrated to be chemically
protected (Brown, 1985; Trigo et al., 1996). This is surprising as

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aposematic colour patterns, rather than inconspicuousness, are
more common in toxic and unpalatable prey (Mappes et al., 2005;
Poulton, 1890; Ruxton, Sherratt, & Speed, 20 04). In fact, conspic‐
uousness is positively correlated with toxicity or unpalatability
in some species and can thus be an honest indicator of prey de‐
fences (Arenas, Walter, & Stevens, 2015; Blount, Speed, Ruxton,
& Stephens, 2009; Maan & Cummings, 2012; Prudic, Skemp, &
Papaj, 20 07; Sherrat t & Beatty, 2003). Moreover, predators learn
more quickly to avoid unpalatable prey when colours are more
conspicuous (Gittleman & Harvey, 1980; Lindstrom, Alatalo,
Mappes, Riipi, & Vertainen, 1999). 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 exist‐
ence of mimicry rings of transparent clearwing butterflies remains
unexplained, as this is usually the result of convergence of warn‐
ing signals promoted by the positive frequency‐dependent selec‐
tion exerted by predators (Willmott et al., 2017). Alternatively,
if defences are costly, prey may invest in either visual or chemi‐
cal defences (Darst, Cummings, & Cannatella, 2006; Speed &
Ruxton, 2007; Wang, 2011), as such options have been shown to
afford equivalent avoidance by predators (Darst et al., 2006). In
which case, transparency should instead be associated with an
increase in unpalatability. This relationship between transpar‐
ency and chemical defences in clearwing butterflies remains to
be explored.
Alternatively, transparency may lower detection and function
as a primar y defence, with aposematism taking over as a second‐
ary defence if the prey is detected. Indeed, transparent butterflies
were not completely cr yptic 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
butter flies. Moreover, humans appear to learn to detect and per‐
haps remember common elements between the more transpar‐
ent species, which might be the result of a search image. As such,
Ithomiini butter flies may be cryptic from afar, but perceived as
conspicuous from up close. The combination of crypsis and con‐
spicuousness has also been shown for other defended prey (Järvi,
Sillén‐Tullberg, & Wiklund, 1981; Sillén‐Tullberg, 1985). For exam‐
ple, toxic salamanders of the genus Tar icha are generally cryptic,
only revealing their warning coloured underbelly when threatened
(Johnson & Brodie, 1975). 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 ar‐
tificial moths (Stevens & Cuthill, 2006). Furthermore, pigment ary
or structurally produced opaque colours, such as the white band
in B. seba, may also enhance but terfly detection. This suggest s,
as do our results and the occurrence of co‐mimics in natural hab‐
itats, that these butterflies may reduce the cost of conspicuous‐
ness 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 protec‐
tion against birds such as adult kingbirds (Tyrannus melancholicus,
Pinheiro, 1996), which are able to tolerate their chemical defences.
Indeed, both theoretical (Endler & Mappes, 20 04) and experimental
(Mappes, Kokko, Ojala, & Lindström, 2014; Valkonen et al., 2012)
studies have shown that weak warning signals (not overtly conspic‐
uous) can evolve and be maintained in communities where preda‐
tors var y in their probability of attacking defended prey. Larvae of
Dryas iulia butter flies, pine sawfly larvae (e.g., Neodiprion sertifer),
and shield bugs (Acanthosomatidae, Heteroptera) are only a few
of the examples that exist of unpalatable species that display weak
visual warning signals (Endler & Mappes, 2004). As in the polymor‐
phic poison frog Oophaga granulifera, clearwing species may reflect
a continuum between aposematism and cr ypsis, 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 sen‐
sitivities to chemical compounds (Willink, Brenes‐Mora, Bolaños, &
Pröhl, 2013). A thorough characterization of unpalatability, micro‐
habitat and predator communities would be useful in better under‐
standing conditions that promote the evolution of transparency.
5 | 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 terres‐
trial aposematic organisms. We show for the first time that transpar‐
ency results in the reduction of detec tability 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 recognized as warning signals. Future studies exploring the
efficiency of combining transparency and warning signals in de‐
creasing predation risk will further contribute to our understanding
of the evolution of cryptic elements in aposematic prey.
ACKNOWLEDGEMENTS
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 permit s in Peru (collecting and expor tation permit 0 02‐2015‐
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 butter fly colour pattern patch and for Ithomiini pic‐
tures, Konnevesi Research Station, which provided the facilities used
for bird experiment s, 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 programme (ANR-16-CE02-0012)
and the Human Frontier Science Program grant (RGP 0014/2016).

|
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AUTHORS’ CONTRIBUTIONS
D.G., M.E., J.M. and M.A. designed the study; M.E., M.M. and D.G.
collected the butterfly samples; M.A., S.G., O.N., M.E. and J.M.
performed the experiments; D.G. and C.D. did the optical measure‐
ments; M.A., D.G. and M.E. analysed the data; M.A., D.G., M.M.,
M.E., O.N., S.G. and J.M. wrote the manuscript. Authors have none
conflict of interest to declare.
DATA ACCESSIBILITY
Data available from the Dryad Digital Repositor y https://doi.
org/10.5061/dryad.17pk7v8 (Arias et al., 2019).
ORCID
Mónica Arias https://orcid.org/0000‐0003‐1331‐2604
Johanna Mappes https://orcid.org/0000‐0002‐1117‐5629
Swanne Gordon https://orcid.org/0000‐0002‐9840‐725X
Melanie McClure https://orcid.org/0000‐0003‐3590‐4002
Ossi Nokelainen https://orcid.org/0000‐0002‐0278‐6698
Doris Gomez https://orcid.org/0000‐0002‐9144‐3426
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SUPPORTING INFORMATION
Additional supporting information may be found online in the
Suppor ting Information section at the end of the article.
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Transparency reduces predator detection in mimetic clearwing
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