Abstract Within-species colour variation is widespread among animals. Understanding how this arises can elucidate evolutionary mechanisms, such as those underlying reproductive isolation and speciation. Here, we investigated whether five island populations of Aegean wall lizards (Podarcis erhardii) have more effective camouflage against their own (local) island substrates than against other (non-local) island substrates to avian predators, and whether this was linked to island differences in substrate appearance. We also investigated whether degree of local substrate matching varied among island populations and between sexes. In most populations, both sexes were better matched against local backgrounds than against non-local backgrounds, particularly in terms of luminance (perceived lightness), which usually occurred when local and non-local backgrounds were different in appearance. This was found even between island populations that historically had a land connection and in populations that have been isolated relatively recently, suggesting that isolation in these distinct island environments has been sufficient to cause enhanced local background matching, sometimes on a rapid evolutionary time-scale. However, heightened local matching was poorer in populations inhabiting more variable and unstable environments with a prolonged history of volcanic activity. Overall, these results show that lizard coloration is tuned to provide camouflage in local environments, either due to genetic adaptation or changes during development. Yet, the occurrence and extent of selection for local matching may depend on specific conditions associated with local ecology and biogeographic history. These results emphasize how anti-predator adaptations to different environments can drive divergence within a species, which may contribute to reproductive isolation among populations and lead to ecological speciation.

Citation: Marshall KLA, Philpot KE, Damas-Moreira I, Stevens M (2015) Intraspecific Colour Variation among Lizards in Distinct Island Environments Enhances Local Camouflage. PLoS ONE 10(9): e0135241. https://doi.org/10.1371/journal.pone.0135241 Editor: Daniel Osorio, University of Sussex, UNITED KINGDOM Received: May 15, 2015; Accepted: July 20, 2015; Published: September 15, 2015 Copyright: © 2015 Marshall et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Data Availability: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by a Biotechnology and Biological Sciences Research Council studentship (www.BBSRC.com), the British Herpetological Society (www.thebhs.org), the Cambridge Philosophical Society (http://www.cambridgephilosophicalsociety.org) and Magdalene College, Cambridge (to KLAM), and a Biotechnology and Biological Sciences Research Council and David Philips Research Fellowship (grant number BB/G022887/1) to MS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Intraspecific variation of colour patterns is a long studied phenomenon in animals, and has profoundly contributed to our understanding of evolutionary processes. Classic experiments have shown that natural selection can drive variation in coloration among populations to optimize survival against predators in varying local environments ([1–3]). It is now understood that adaptation to local environments can cause reproductive isolation between divergent populations and potentially lead to ecological speciation (reviewed in [4–6]). Various studies on mice and lizards have shown that divergent populations undergo selection to better match local backgrounds for camouflage, and that this involves mutations of key genes and is influenced by levels of gene flow (e.g. [7–11]). However, until recently, relatively few studies have based their conclusions on the visual systems of potential predators, such as hunting birds (but see [12, 13]). As the avian predator visual system and perception of background matching camouflage differs from that of humans (for example, birds can see ultraviolet light and probably a greater range of colours; [14, 15]), it is important to determine whether predators perceive divergence in camouflage among local environments (e.g. [16, 17]). As local adaptation for camouflage should involve selection for coloration that matches associated backgrounds, it can be inferred only if local background environments are different. This is because coloration could become variable through other processes, such as genetic drift, even among similar environments. Variation may also arise owing to phenotypic or developmental plasticity, whereby individuals can change colour during their lifetimes (e.g. [18–21]). Consequently, it is important to determine how predator-perceived camouflage and visual backgrounds vary among populations in order to understand local adaptation and its role in intraspecific divergence. The Aegean wall lizard (Podarcis erhardii; [22]) is a valuable model species for this type of study because different island populations exhibit clearly visible colour variation (to humans) among ostensibly distinct island environments (see Fig 1). These island populations have been historically categorised as different subspecies due to their varying appearance ([23]; but see more recent molecular analyses in [24, 25]) and show genetic diversity between isolated islands with no recent common history ([26]). The variable environments of the islands are widely thought to have driven the apparent phenotypic and genetic diversification among the inhabitant P. erhardii populations (via the equivalent of a sustained bottleneck) dating from their time of separation ([25–28]), although this has not been formally tested. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 1. Example images of Aegean wall lizards (Podarcis erhardii) and their typical island habitats. Images depict typical natural habitat and dorsal coloration of males (left image) and females (right image) in each focal island population (Nea Kameni, Folegandros, Santorini, Skopelos and Syros). Images were obtained in the field by the authors. https://doi.org/10.1371/journal.pone.0135241.g001 Studying P. erhardii in this island system differs from a range of previous studies focusing on the adaptive significance of colour polymorphisms among sympatric lizard populations (e.g. [7, 9, 13, 29]). In particular, examining colour variation between isolated island populations can potentially reveal how distinct ecological factors, as well as time of isolation and evolutionary history (e.g. historical geographical connections), may drive differential local adaptation of camouflage and thus further clarify the selective conditions under which it can occur. Specifically, island populations of P. erhardii vary in habitat type (e.g. geological characteristics and habitat composition), predation risk (i.e., number of resident avian predators), time of isolation (ranging from >200,000 to 400 years), and potential amounts of gene flow from other island populations depending on historical geographic connections and amounts of boat traffic (see Table 1; [25–28, 30–32]). For example, the Nea Kameni islet has been formed from intermittent volcanic eruptions over the last 400 years ([33]) and inhabitant P. erhardii rest on homogeneous black lava rock backgrounds, whereas the land bridge island of Syros has been isolated for over 13,000 years and lizards rest on various metamorphic rock backgrounds (Table 1; [27, 34]). PPT PowerPoint slide

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larger image TIFF original image Download: Table 1. Differences in the environments and evolutionary histories of the five focal islands inhabited by populations of Aegean wall lizards (Podarcis erhardii). https://doi.org/10.1371/journal.pone.0135241.t001 A recent study showed that P. erhardii island populations are tuned to their local island environments to be conspicuous to conspecifics while also being camouflaged against avian predators ([36]). Specifically, P. erhardii coloration was found to be less perceptible by avian predators than by conspecifics on all three focal islands. Moreover, dorsal regions (upper and lower backs) exposed to aerially hunting birds were relatively more camouflaged against local rock backgrounds compared to less exposed lateral (flank) regions, and this was more evident on some islands compared to others. In addition, on all islands, sexually competing males showed inferior camouflage on their upper backs compared to that of females. Given the apparent environmental variation among the islands, these findings suggest that P. erhardii have adapted to their local environments to enhance dorsal camouflage against avian predators, and also that degree of local adaptation varies among islands, and between sexes and upper and lower backs. However, this study did not test whether the backgrounds and lizard populations were variable among islands, or whether lizards were more camouflaged against their local island backgrounds compared to that of different islands. Therefore, in the current study we directly test these hypotheses, and also consider how different ecological selection pressures (e.g. predation risk) and evolutionary history (e.g. time of island isolation) has influenced local selection on dorsal camouflage in each population (Table 1). It is likely that island populations of P. erhardii have adapted to be camouflaged in their local environments for several reasons. The populations are under considerable though variable threat from avian predators (i.e., raptors and corvids), particularly as they tend to bask on rocks in open environments where they are often visible to hunting birds, as in other lizards (Table 1; [31, 37–39]; pers. observations). Therefore, it is likely that selection has favoured colour matching against local rock backgrounds to reduce detection by avian predators. However, the variable threat from avian predators among the islands may affect degree of local adaptation for camouflage, as shown in past work on other lizards (e.g. [13, 29, 40]). Additionally, various studies have shown that there is relatively little gene flow (i.e., via over-water dispersal) between most island populations ([26–28, 41]), which is similar to other lizards showing phenotypic diversification among recently isolated islands where dispersal across seawater is also extremely rare ([42]). Thus, most of the current P. erhardii populations are highly likely to be descendants of the original colonists that have adapted to their native environments since they became isolated from neighbouring landmasses. This is substantiated by past work showing genetic adaptation of lizard coloration to environments that have existed for only 6,000 years (e.g. [10, 29]), which is a comparable age to most island environments considered in the current study (Table 1; [27]). We investigated whether P. erhardii populations are more camouflaged on their local island than on non-local islands to avian predators, and whether this was associated with lizard and environmental (substrate) differences in appearance between local and non-local islands. We predicted that there would be differences in coloration between P. erhardii island populations and between their rock background environments, and that lizards would show better camouflage against local backgrounds than against non-local backgrounds to avian predators. We also predicted that there would be variation in degree of camouflage among islands with different evolutionary histories and environments, and between sexes and body regions.

Materials and Methods Study species and sites The Aegean wall lizard (Podarcis erhardii) is a diurnal, small lacertid distributed across most of the South Balkans and widespread throughout many Aegean islands where it is found in all ecosystems ([43, 44]). We conducted field research during the activity season (April-July; [45]) between 2012–2014 on five Aegean islands with three different inhabitant subspecies (classified based on appearance; [23]): P.e. naxensis (Folegandros [36°37’ N, 24°54’ E], Santorini [36°25’ N, 25°26’ E] and the neighbouring islet Nea Kameni [36°24’ N, 25°24’ E]; P.e. mykonensis (Syros [37°27’ N, 24°54’ E]); and P.e. ruthveni (Skopelos [39°7’ N, 23°43’ E]). We chose to sample lizards from these islands because they have ostensibly varying coloration and environments, substantial but variable risk from avian predators, and different evolutionary histories (see Fig 1; Table 1). Ethics statement P. erhardii is listed as a species of ‘least concern’ under the IUCN Red List classification, so it is not an endangered or protected species ([46]). We conducted field research with permission from the Greek Ministry of Environment (permit numbers: 166648/356 and 107222/707). These permits were granted for all study sites, and all land used for fieldwork was publicly accessible. The permits also gave us permission to sample lizard coloration using all methods as described. Sampling methods were non-invasive, involving photography of lizards in situ. However, in one study site (Nea Kameni islet) lizards were captured for photography, because this islet has a low population density and we had restricted access with a limited amount of time to obtain an appropriate sample size. Lizards were caught using a noose consisting of a slipknot attached to a telescopic pole, which is widely used by herpetologists as a non-harmful capture method (e.g. [47]). Nonetheless, we endeavoured to release the lizard from the noose as quickly as possible after capture, which was usually within five seconds and never more than 10 seconds. All researchers had prior experience using this capture method and with handling lizards. After capture, lizards were immediately photographed, marked with a small amount of non-toxic (water-based), inconspicuous (dark green) paint (Montana, Heidelberg, Germany) on the vertical surface of their hind leg so as not to alter dorsal camouflage, and then released at the original (marked) point of capture. During capture, we ensured that we handled lizards in a way that minimized the likelihood of tail autotomy and aimed to release them as soon as possible, which was typically within five minutes. Lizards were never harmed and all showed normal behaviour after release. We captured the minimum number of lizards necessary to rigorously test our hypotheses (N = 32; 19 males, 13 females). All protocols conformed to the policies and requirements of the ethics committee of our institution. In situ photography We used digital imaging to sample lizards and their corresponding background coloration, because it has various advantages over alternative spectrometry methods (see [36, 48]). As in ([36]), during in situ photography we took images of stationary lizards and their corresponding natural backgrounds with a Fujifilm IS Pro ultraviolet (UV)-sensitive digital camera with a quartz CoastalOpt UV lens (Coastal Optical Systems), fitted with a UV and infrared (IR) blocking filter for photographs in the human-visible spectrum (Baader UV/IR Cut filter; transmitting between 400 and 700 nm), and with a UV pass filter (Baader U filter; transmitting between 300 and 400nm) for UV images. After the photographed lizard had fled, we took human-visible and UV images of a SpectralonTM grey reflectance standard (Labsphere, Congleton, UK), which reflects light equally at 40 per cent between 300 and 750 nm. Following this ‘sequential method’ as used in past work ([49, 50]), images of the standard were taken at the same distance, with the same camera settings, and in the same location and light conditions, as the photographed lizard. This enabled us to undertake the required standardization for ambient light conditions (i.e. image linearization and RGB-equalization; see below) (see [48]). Light conditions rarely changed in the typical 2–3 minutes between photographing the lizard and the standard. However, on the few occasions when this did occur (e.g. clouds passed over the sun), we re-photographed the standard once the lighting had returned to normal. We endeavoured to photograph most lizards (and hence standards) in full sunlight. In the few cases where shadows partly obscured the image, our selections avoided shadowed areas and only measured parts of the lizards, backgrounds and standards that were in sunlight. We recorded photographed lizards’ locations using a Garmin eTrex GPS device (Schauffhausen, Switzerland) and marked it with coloured tape to indicate sex and life-stage ([43]). We confirmed these estimations were 99% reliable by comparing estimated (from photographs) and observed sex and life-stages from captured lizards (see [36]). We avoided pseudoreplication by never repeating photography of a lizard of the same sex within the same home range (i.e. within 10 m) (see [36, 51]). On the Nea Kameni islet we captured lizards for photography. Capture location was marked with coloured tape for subsequent release. To minimise stress and to avoid any colour fading through decreases in body temperature, which can sometimes arise during long-lasting stressful situations in lizards (e.g. [52, 53]), lizards were photographed within five minutes after capture. The grey reflectance standard was photographed and lizards were marked as described previously. Observations and other mark re-capture studies showed that marks lasted for at least three weeks (K. Marshall and I. Damas-Moreira, unpublished data). After allowing the paint to dry, lizards were released at the marked capture location. Capture attempts were not made within 10m of the same location again. To ensure that lizards would be subsequently compared to their natural corresponding backgrounds, each (marked) background that each lizard had been resting on at the point of capture was photographed with the grey standard in the image. Image analysis and visual modelling We followed methods previously described in ([36]). Human-visible and UV images of lizards and their backgrounds were linearized with respect to light intensity and transformed to reflectance (RGB-equalized; see [48]). Any images that were overexposed and/or could not be RGB-equalized were discarded from the analysis. We then used a mapping process based on the spectral sensitivity of our camera’s sensors (derived prior to photography; see [36]) to convert the images to correspond to avian predicted photon catch cone values ([48]). This is highly accurate compared to spectrometry-derived estimates of animal photon catch values (see [54–56]). We converted the aligned images from camera colour space to the relative photon catches of an avian predator’s longwave (LW), mediumwave (MW), shortwave (SW) and UV sensitive cone photoreceptors using the spectral sensitivity of a peafowl (Pavo cristatus; [14]). We also mapped the images to peafowl double cone photoreceptors ([57]) to represent achromatic (luminance) avian perception ([58, 59]). The peafowl visual system is often used as a representative of the violet-sensitive (VS) class of colour vision in birds ([60, 61]). The VS-system is typical of the predatory birds that hunt Podarcis lizards and other lacertids in Europe (i.e. raptors and corvids; [31, 37, 62]), which has recently been supported by molecular studies tracing the evolution of ultraviolet vision in birds ([63]). Although some birds with ultraviolet-sensitive (UVS) vision might also prey on P. erhardii (e.g. gulls and some Turdidae (thrush) species that are present in our study sites; [63–66]), we considered VS-sensitive raptors and corvids as the more important avian predators of adult lizards in Europe ([31, 37, 62, 63, 67]). Indeed, we often observed hooded crows (Corvus cornix) and a variety of raptors (e.g. buzzards, Buteo buteo; common kestrels, Falco tinnunculus; Eleonora’s falcon, Falco eleonorae) hunting in our study sites, while gulls and thrushes were rarely seen. Calibrations were performed in MATLAB v. R2011b (The MathWorks, Inc., MA, USA) using self-written programs. Both calibrations were restricted to the 300–700 nm range, which encompasses most of the visual spectrum of diurnal birds ([61]). LW, MW, SW and UV photon catches of each lizard and their corresponding background (i.e., the background we photographed the lizard resting on) were extracted from the calibrated images in ImageJ using the selection tool. Selection of a particular area in the image generated an average photon catch value for that patch. Background selections were limited to rock backgrounds because Podarcis lizards most frequently bask on rocks (given their good thermal quality) where they are potentially visible to aerially hunting predators in open environments (e.g. [38, 39, 68]; pers. observations). We randomly selected areas of rock that were adjacent to the photographed lizard, so that the selection touched but did not overlap it, and avoided areas of lichen and moss. Lizards sometimes rested on rock backgrounds that consisted of different patches of colour patterns, although we could usually identify the predominant (largest) patch from which we made our background selection. However, in some images we could not identify a predominant background patch. In these cases, we made up to three selections of different patches and averaged the photon catches across patches as a representative substrate colour. Lizard selections were made from two dorsal body regions (posterior lower and anterior upper backs). The average photon catch value extracted by these selections included dorsal patterning, which is particularly extensive in males (see Fig 1). We assumed that this would depict how lizards appear to birds hunting from a distance, especially given that the lizards are small (i.e. ≈7cm snout-to-vent length; [43]). However, we acknowledge that at close range patterning might be more distinguishable by birds and serve another function (e.g. disruptive camouflage; [69]). Separate selections of lower and upper backs were taken due to observed colour differences between these regions, and selection of these body regions was standardized across all images. Specifically, upper back selections were taken under the base of the head and lower backs selections were taken above the base of the tail (see [36]). A total of 295 adult lizards and their corresponding rock backgrounds were sampled from the five focal island populations (Folegandros = 100; 52 males, 48 females, Syros = 49; 34 males, 15 females; Santorini = 58; 34 males, 24 females; Skopelos = 56; 29 males, 27 females; and Nea Kameni = 32; 19 males, 13 females). Island differences in lizards and backgrounds We first aimed to ascertain any differences in colour and luminance among the island lizard populations and their rock background environments. We calculated a variety of metrics to use in our analysis. Saturation (the amount of a certain colour compared to white light) refers to the Euclidian distance a colour patch is in a tetrahedral colour space from the achromatic (grey) point, with larger values representing more saturated colours (see [16]). We also quantified the type of colour (hue). This was based on a ratio of the different colour channels present (i.e., LW, MW, SW and UV) to represent relative photoreceptor stimulation by the colour patch spanning different parts of the light spectrum and receptor types ([55, 70–72]). This followed an approach developed in past work, which aims to quantify colour so that it broadly reflects how colour is encoded by an animal’s visual system (see [56, 71, 72]). Specifically, we used a ratio based on the concept that colour is encoded using opponent (antagonistic) colour channels or neural pathways, such as the ‘red-green’ and ‘blue-yellow’ pathways in trichromatic primates ([59, 73–75]). Since it is unknown which specific opponent colour channels birds have, we identified biologically relevant putative colour channels by performing a principle component analysis (PCA) on a covariance matrix of the standardized cone photon catches derived from our avian visual model ([71]). This enabled us to determine one or two colour channels that best described the variation in colours existing among lizards ([56, 72]). Two PCs were extracted and together explained almost all of the variance in lizard and background coloration (lower backs = 95.2%; upper backs = 93.6%; backgrounds = 97.8%). PC1 showed highest eigenvalue loadings on the LW and SW channels, and PC2 showed highest loadings on the MW and UV channels. Therefore, we used the following standardized ratio to identify perceived differences in the identified opponent colour channels ((LW+SW)-(UV+MW)/UV+SW+MW+LW); see [69, 71]). Consequently, this ratio would reflect differences between perceived ‘purple’ and ‘green-UV’ coloration. Finally, for a measure of luminance (i.e. perceived lightness), we used standardized double cone values derived from the avian visual model ([57–59]). Degree of island differences in lizards and backgrounds In addition to calculating the hue, saturation and luminance metrics separately, we sought more insight into the divergence between islands by determining the extent of the perceived differences in lizards and their rock backgrounds, and to understand perceptual differences in colour accounting for both hue and saturation together. To do this, we measured the degree of contrast between each island population (i.e., lizards vs. lizards) to identify how similar lizards were to members of their local population and to members of non-local populations. We also measured the degree of contrast between the measured backgrounds (i.e., backgrounds vs. backgrounds) to identify how similar rock backgrounds were to those of the local island and to those of non-local islands. We quantified degree of colour (chromatic) contrast according to the widely used log form of a receptor noise model ([76]), which predicts visual discrimination abilities in observers. We also quantified luminance contrasts using a version of the model based on achromatic differences using peafowl double cones ([77]). To account for receptor noise, we used a Weber fraction value of 0.05 for the most frequent cone type based on data in other vertebrates ([76, 78]). We used relative proportions of cone types in the peafowl retina to calculate avian predator-perceived chromatic contrast (LW = 0.92, MW = 1.00, SW = 0.81, UV = 0.54; [14]). The degree of chromatic and achromatic contrast generated from these models is expressed as a measure of contrast called “just-noticeable-difference” (JND), which predicts discrimination behaviour in observers. Generally, JND values increasing above 3.00 indicate increasingly improved discrimination ([77]). We compared each lizard with every lizard belonging to all island populations, including to its local population and to all four non-local populations. We performed the same comparisons with the backgrounds to determine whether differences in visual backgrounds were large enough to be perceptibly different to avian predators, and thus cause changes in lizard appearance to match them on each island. To control for sexual dichromatism in lizards’ dorsal coloration and variation in coloration between upper and lower backs ([36]), lizards were compared with only members of the same sex and only equivalent body regions were compared. After obtaining JNDs for all comparisons, we calculated the average JND (i.e., degree of contrast) for each lizard and each background to its local island and to each of the four non-local islands. Local adaptation of camouflage Lastly, we determined to what extent the lizard populations matched different (local and non-local) rock backgrounds for background-matching camouflage. To ascertain any differences in camouflage among islands, we used the receptor noise model described above ([76]) to compare each lizard with all local island backgrounds and with all non-local backgrounds from the four different islands. We calculated the average JND for each lizard to ascertain how well they matched local and non-local island backgrounds. Predictions and statistical analyses Normality tests and residuals analysis showed a normal distribution of hue, but the saturation, luminance and JND data were not normally distributed with positively skewed distributions. Therefore, we transformed this data to normality using a logarithmic transformation and used the transformed data in all statistical analyses. However, to illustrate and describe our results, we report raw (back-transformed) JND data in figures. All statistical analyses were conducted in SPSS® (v20). First, we predicted that coloration and luminance of lizards and their rock backgrounds would differ among islands and be more similar to those on local islands as opposed to those on non-local islands, particularly between islands that have never shared a historical geographical connection and that have different subspecies (see Table 1). We also expected lizards to vary between sexes and body regions due to sexual dichromatism ([36]). To address these predictions, we first conducted a series of general linear models (GLMs) on the colour (hue and saturation) and luminance metrics. To test for differences among island backgrounds, we carried out univariate GLMs with island (Folegandros, Nea Kameni, Santorini, Skopelos and Syros) included as the between-subjects factor. To test for differences in lizard populations, we conducted mixed GLMs with island and sex as between-subjects factors and body region (upper and lower backs) as the within-subjects factor. We also conducted mixed GLMs on the chromatic and achromatic lizards vs. lizards JND data (tests 1 and 2) and backgrounds vs. backgrounds JND data (tests 3 and 4). In tests 1 and 2, island and sex were included as between-subjects factors and body region (upper and lower backs) and ‘compared island’ (i.e., which island the compared lizard belonged to) were included as the within-subjects factors. The same variables were included in tests 3 and 4, with the exception of body region and sex. Second, we predicted that lizards would match their local backgrounds better than backgrounds on other islands for camouflage against avian predators, and that this would be accompanied by environmental (rock background) differences between islands. In addition, we predicted that this effect would be weaker in populations that have been isolated for relatively shorter evolutionary periods and have ecological characteristics that reduce the need for camouflage, such as lower levels of risk from avian predators (see Table 1). We also predicted that females would show better local adaptation for camouflage compared to sexually competing males, and that lower backs would show enhanced local camouflage compared to sexually dichromatic upper backs. To address these predictions, we conducted two mixed GLMs, one testing chromatic contrast (test 5) and the other analysing achromatic contrast (test 6) against backgrounds (JND). In both GLMs, we included sex and island lizard population as between-subjects factors. The ‘compared island’ backgrounds (i.e. which island background the lizard was compared to) and body region were included as within-subjects factors. In both tests 5 and 6 we specifically looked for a factor interaction between island lizard population and ‘compared island’ background to determine if degree of camouflage (JND) depended on whether lizards were compared with their local island background or with a different (non-local) island background. In all tests we also report any significant effects of sex and body region on lizard differences between islands and degree of local adaptation of camouflage. We report the size of all effects in ETA2 (η2), which can be interpreted as the proportion of variance in the dependent variable that is attributable to each effect. To determine the sources of variation in any significant effects, planned pairwise comparisons were conducted by re-running the GLMs with only the factors of interest included that were relevant to our predictions. Factors with non-significant effects were removed from the analysis before re-running the GLM to obtain the best model. The number of comparisons was limited to the number of “spare” experimental degrees of freedom (n-1), because these are more powerful than conservative, multiple unplanned post hoc comparisons ([79]).

Supporting Information S1 File. Island differences in colour and luminance of Aegean wall lizards (Podarcis erhardii) and their rock background environments. Occurrence of significant differences and effect sizes (ETA-squared [η2]) are shown between island lizard populations (Table A) and between rock backgrounds (Table B) in terms of hue (top row), saturation (middle) and luminance (bottom). Figure A shows variation in dorsal coloration (hue and saturation) and luminance between the island lizard populations (top row) and their rock backgrounds (bottom row). https://doi.org/10.1371/journal.pone.0135241.s001 (DOCX) S2 File. Degree of contrast (JND) between island lizards and between island backgrounds. Occurrence of significant differences and effect sizes (ETA-squared [η2]) are shown from statistical analyses comparing degree of contrast (JND) between island populations of Aegean wall lizards (Podarcis erhardii) and degree of contrast between their rock backgrounds (Table A). Chromatic and luminance JNDs are shown between island lizard populations (Figure A) and between their rock backgrounds (Figure B). https://doi.org/10.1371/journal.pone.0135241.s002 (DOCX) S3 File. Island differences in local camouflage. Showing significant differences and effect sizes (ETA-squared [η2]) comparing degree of local camouflage between different island populations of Aegean wall lizards (Podarcis erhardii) (Table A). Degree of dorsal chromatic and luminance camouflage against local and non-local island backgrounds is shown in Figure A. Body region and sex differences in degree of chromatic camouflage (Figure B) and luminance camouflage (Figure C) are shown against local and non-local island backgrounds. https://doi.org/10.1371/journal.pone.0135241.s003 (DOCX)

Acknowledgments We are grateful to N.B. Davies and to two anonymous referees for comments on previous versions. We thank the Greek Ministry of Environment and P. Pafilis for granting permission to conduct this research. We are grateful to S. Finlay, M. Moore and A. Török for excellent assistance in the field. K.L.A.M. thanks Magdalene College and the Department of Zoology, Cambridge.

Author Contributions Conceived and designed the experiments: KLAM MS. Performed the experiments: KLAM KEP ID-M. Analyzed the data: KLAM. Contributed reagents/materials/analysis tools: KLAM MS. Wrote the paper: KLAM KEP ID-M MS.