Plasticine replica experiment
We conducted plasticine replica experiments using natural avian predators in the mountain forests of Okinawajima Island, Ryukyu Archipelago, Japan. At the field site, S. japonicus boettgeri is active during the day and at night on the ground (Obara 2013; Mochida and Toda, personal observation). We constructed two types of snake replicas using pre-colored plasticine (Sculpy-III; Polyform Products Co., Elk Grove Village, IL, USA) threaded onto an S-shaped wire frame (Brodie 1993). Coral snake replicas were painted black with narrow white transverse bands and black stripes on an orange surface similar to the body color pattern of S. japonicus boettgeri, and the control replicas were plain green, similar to Cyclophiops semicarinatus, which is another prey item of avian predators at the field site (Tanaka and Mori 2000). We verified that the coloration of the replicas did not exhibit appreciable difference in reflectance spectra to that of target snakes using a USB2000+ spectrometer (Ocean Optics, Dunedin, FL, USA) and that there were no snake species showing coloration and pattern similar to replicas except for target snakes. The size of both replicas was 10 mm diameter and 180 mm length. Both types of replica were placed into one location side by side, enabling the avian predators to choose a profitable prey item (Figure S1 in Additional file 1). If the body color pattern of S. japonicus boettgeri functions aposematically, avian predators would avoid predation attempts on replicas of a similar color pattern. The replica pairs were placed on forest floor 0.5 to 1.0 m from edge of mountain roads. We prepared two types of background, an artificial white board and natural backgrounds, as predators might not find them on natural backgrounds if both replicas were too cryptic for them to locate. In one experiment, 25 pairs of replicas were set on each background in an alternating arrangement. Fifty pairs of replicas were divided into three blocks at intervals of 2 km, with each pair set at over 10-m intervals within a block. The number of pairs was from 12 to 20 in each block. After 72 h, the replicas were collected, and the U-shaped or V-shaped bill marks left by avian predators on the replicas were recorded as attacking attempts on them (Brodie 1993; Brodie and Janzen 1995; Valkonen et al. 2011). Multiple marks on one replica were scored as a single predation attempt. The experiments were conducted in November 2012 and June 2013, using a total of 100 pairs of snake replicas. November is the season when the snake-eating buzzard Butastur indicus is temporally abundant at the field site because of its migration from northern areas. Some of them remain there during winter, and then the numbers increase again in early spring until they disappear from the field site by May. Besides the buzzards, there are also a few avian species, which are potential predators on snakes, such as crows and kingfishers, throughout the year. Because some of the replicas were carried away from their original locations in the first experiment (see Results), both sides of the replicas were fixed to the ground in the second experiment.
Color analysis
The chromatic contrasts between the color elements of the snake body and background objects, and between the color elements within a snake body in S. japonicus boettgeri, were analyzed. These chromatic contrasts were compared to those of the two nonvenomous colubrid species, C. semicarinatus and Dinodon semicarinatum, and an ambush venomous pit viper, Ovophis okinavensis, all found on Okinawajima Island and living sympatrically with S. japonicus boettgeri. D. semicarinatum has large black blotches and a reddish white color, and the body coloration of O. okinavensis consists of small dark brown blotches on a light brown color surface (Figure S2 in Additional file 1). The main habitat of these four snake species is the forest floor, which is covered with red clay and fallen leaves. The reflectance spectra were measured of the orange and black color elements of S. japonicus boettgeri (N = 5), a green color element of C. semicarinatus (N = 5), the black and reddish-white color elements of D. semicarinatum (N = 8), and the light and dark brown color elements of O. okinavensis (N = 11). The spectra of the soil and fallen leaves in their habitats were analyzed as background color elements. For each background type, 20 subjects were measured. In these analyses, a USB2000+ spectrometer was used with a DT-Mini-2-GS deuterium and halogen light source (Ocean Optics, Dunedin, FL, USA). The average reflectance of each color element was calculated by computing the means from three replicate spectra measured for each subject for each data point between 300 and 700 nm, expressed relative to a 99% white reflectance standard. Irradiance of their habitats was also measured using a USB2000+ spectrometer with a cosine adapter head. We used LS-1 CAL as the standard reflectance light. In April 2013, we measured the irradiance at three landscape locations in their habitats: the ridge, valley, and the gentle slope of the mountain forest, under full sun conditions between 10:00 and 14:00 h. The average irradiance of the habitat was calculated by computing the means from 10 irradiance data measures for each location for each data point between 300 and 700 nm. To evaluate the chromatic contrasts of the snake body coloration, all color spectra data were mapped in a tetrahedral space, based on environmental irradiance and ultraviolet-sensitive (UVS) or violet-sensitive (VS) visual systems, as birds have two types of color vision (Endler and Mieke 2005). To calculate the chromatic contrast in the tetrahedral space, the coloration, represented by the color covering the largest surface area of each species, was set as the base color element (S. japonicus boettgeri; orange, C. semicarinatus; green, D. semicarinatum; black, and O. okinavensis; light brown). Based on these color elements, chromatic contrasts were calculated using formula (22) in Endler and Mieke (2005). We also calculated these contrasts based on a black color element that covers the second largest area of its body surface in S. japonicus boettgeri. C. semicarinatus shows plain green coloration; therefore, we did not calculate intrabody contrast in this species. Animals were handled and experiments were conducted in accord with the Guideline for Animal Experiments in University of the Ryukyus.