Rapid evolution of a Batesian mimicry trait in a butterfly responding to arrival of a new model

Batesian mimicry, a phenomenon in which harmless organisms resemble harmful or unpalatable species, has been extensively studied in evolutionary biology. Model species may differ from population to population of a single mimetic species, so different predation pressures might have driven micro-evolution towards better mimicry among regions. However, there is scant direct evidence of micro-evolutionary change over time in mimicry traits. Papilio polytes shows female-limited Batesian mimicry. On Okinawa, one mimicry model is Pachliopta aristolochiae, which was not present on the island until 1993. In P. polytes, the size of the hind-wing white spot, a mimetic trait, is maternally heritable. Among specimens collected between 1961 and 2016, the average white spot size was unchanged before the model’s arrival but has rapidly increased since then. However, white spot size showed greater variance after the model’s establishment than before. This suggests that before 1993, white spot size in this population was not selectively neutral but was an adaptive trait for mimicking an unpalatable native, Byasa alcinous, which looks like P. aristolochiae apart from the latter’s hind-wing white spot. Thus, some females switched their model to the new one after its arrival.

by a conspicuous white spot in the centre of the hind wing surrounded by several small red spots ( Fig. 1D-F). These various wing colour patterns suggest that regionally different predation pressures related to differences in model morphology drove micro-evolution towards better mimicry in each region.
The Ryukyu Archipelago, situated in the subtropical region of Japan, lies at the northern end of the species distribution of P. polytes. Forma polytes is the only mimetic morph found on the Ryukyus. Historically, its putative 'white spot model' was not distributed in Japan. The distribution of P. aristolochiae has been expanding recently, however, and this species began establishing populations on some islands of the Ryukyus in the 1960s. Uesugi 13 observed a rapid increase in the proportion of mimetic females after the arrival of the model on Miyako Island, in what can be regarded as a direct observation of the evolutionary process of Batesian mimicry 13,14 . Uesugi 13 also noted that the size of the hind-wing white spot of mimetic females was highly variable in the Ryukyus, and that the average spot size was larger on islands where the model P. aristolochiae was distributed than on islands where it was absent, including Okinawa (the main island of the Ryukyus). Two years after Uesugi's report, in 1993, P. aristolochiae established a population on Okinawa 15 (the distribution record of Clarke and Sheppard 3 has a mistake: P. aristolochiae was not distributed on Okinawa in 1972). This provides us with another fortunate opportunity to directly observe the presumable micro-evolutionary change in the mimetic wing colour patterns.
We hypothesized that because of the changed predation pressure due to the immigration of the white spot model, natural selection (directional and stabilizing selection) would have driven the wing colour patterns of f. polytes to more closely resemble the model from the former selectively neutral state in the absence of the model. To test this hypothesis, we first estimated the heritability levels of the red and white spot patterns of f. polytes in the field population. The presence of heritable variation in those quantitative traits enables micro-evolution in response to natural selection 16 . Second, using specimens in museum and private collections and some captured by us, we analysed quantitative changes in the size of the white spot over more than five decades in the field populations of Okinawa and other islands (the Miyako and Yaeyama groups) of the Ryukyus.

Results
Estimation of heritability of mimicry traits. The broad-sense heritability (H 2 ) of the white spot size (relative to the hind-wing area) was significant, whereas that of the red spot size was not significant ( Table 1). The white spot size was inherited only maternally, with heritability exceeding 1 (Table 1). There was no significant genetic correlation between the white and red spot sizes (maternal r = 0.152, P = 0.134; paternal r = 0.065, P = 0.351). These results indicate that the white spot size in the mimetic females can change in response to natural selection. Change in spot size over time. Using specimens in museum and private collections and some captured by us, we analysed changes in the size of the white spot over the last 55 years in the field population on Okinawa. The results showed that the white spot size increased during that period (Fig. 2). It did not change between 1961 and 1991, when the model was absent (regression coefficient b = −0.005, P = 0.819), but it increased from 1994 to 2016, after the model's arrival in 1993 (b = 0.045, P = 0.031). Over time, the white spot size approached the model's range ( Fig. 2A). The variance of the white spot size was greater after 1993 than before (F = 0.56, df = 35, 331, P = 0.044, Fig. 2A).
In the Yaeyama and Miyako island groups, where the model arrived earlier (in 1968 and 1975, respectively), the white spot size seemed to increased over the last 61 years (1955-2016, b = 0.0284, P = 0.145). Remarkably, the timing seemed to differ from that on Okinawa, with a more prominent increase in size before 1992 (b = 0.111, P = 0.172) than after 1993 (b = 0.006, P = 0.885), although neither was significant (Fig. 2B). During the 1980s, the white spot size was larger in the Yaeyama and Miyako island groups than on Okinawa (Student's t-test, t = 3.79, P < 0.01). The spot size of f. polytes in the Yaeyamas and the Miyakos now (in 2016) greatly overlaps with that of the model (Fig. 2B).

Discussion
Batesian mimicry has been regarded as a textbook case for natural selection theory 5,7 . Our results confirm this assertion with real-time micro-evolution data for the following reasons. We clearly showed that the white spot size of Papilio polytes f. polytes on Okinawa changed rapidly to better mimic the model Pachliopta aristolochiae after the model's arrival. A similar change occurred earlier on the Miyako and Yaeyama islands, where the model arrived earlier. These results strongly suggest that the observed change in wing coloration of f. polytes was caused by the arrival of the model, not by an environmental change. Furthermore, there is experimental evidence that birds on Miyako Island that fed on P. aristolochiae started to avoid P. polytes f. polytes 13 . Thus, f. polytes in the Ryukyus really mimics P. aristolochiae.
However, the increased variance of the white spot size after 1993 might contradict our above scenario, because we predicted reduced variance after the model's establishment, owing to presumable stabilizing selection favouring better mimicry. One explanation is that it is an artefact due to the small sample size before 1992: we might have failed to sample outliers before the model's arrival just by chance. However, we consider this to be unlikely,  because the white spot size variation showed a good fit to a normal distribution, where outliers might be less important. Another explanation is that the mimetic trait of P. polytes on Okinawa before the arrival of P. aristolochiae was not a neutral trait but was an adaptive trait mimicking another model. In 1991, Uesugi 17 assumed that the model of f. polytes on Okinawa was the unpalatable butterfly Byasa alcinous, a native to the island. The wing colour pattern of P. aristolochiae (Fig. 1G) closely resembles that of B. alcinous (Fig. 1H) if the hind-wing white spot of P. aristolochiae is ignored. This explanation is consistent with the fact that before the arrival of P. aristolochiae, mimetic females of P. polytes had smaller white spots on average (Fig. 2). Thus, some of the mimetic females might have switched their model to P. aristolochiae. The increased variance of the white spot size of mimetic P. polytes after the arrival of P. aristolochiae can be explained by disruptive selection due to the presence of two models, one with and the other without a white spot. This new scenario still leaves one question, namely why most mimetic females before the arrival of P. aristolochiae still had the white spot, even if it was smaller. In other words, why did they not exhibit a phenotype like another mimetic form found in the tropics, f. theseus, which totally lacks the white spot? To answer this question, we need to ascertain how long P. polytes populations have inhabited Okinawa. Furthermore, studies are needed to elucidate what genetic and developmental changes underlie the micro-evolution described above. The female-limited Batesian mimicry in P. polytes is coded by the autosomal doublesex supergene 10, 12 . However, our analysis indicates that the white spot size is only maternally inherited, and thus the white spot size variation in the studied population is not controlled by an autosomal gene. So a gene that controls white spot size may exist on the W sex chromosome (lepidopterans have ZW sex determination, in which females are heterozygous) or in organelle genomes. The result that the estimated heritability of the white spot size is >1 supports this idea. The grandmother-granddaughter regression model we used assumes autosomal gene control. The alternative assumption of W chromosome or organelle gene control would make the true heritability 1/4 of the values shown in the Results.
This study provides direct evidence of rapid micro-evolution of a mimetic trait in response to community changes due to the extension of the distribution of a new model. This is the first study to directly observe the theoretical process of the evolution of Batesian mimicry, that is, local adaptation to a mimetic trait to better resemble a new local model.

Methods
Quantification of mimicry traits. A wing of each butterfly (ventral view) was photographed with a digital camera (Nikon D7000, Nikon Corporation, Tokyo, Japan) mounted 50 cm directly above the specimen under invariant light (Fig. 3A). The left wing was used unless it was damaged. We analysed the hind-wing area below a horizontal line (Fig. 3B) demarcated as follows. We defined landmark 1 as the intersection of the second anterior cubitus vein (the border between space 1b and space 2) and the border of the cell on the hind wing, and landmark 2 as the tip of the vein on the tail of the hind wing. The vertical line connecting landmarks 1 and 2 was rotated so that it was vertical in Adobe Photoshop CS5 (Adobe Systems Inc., San Jose, CA, USA). Then a horizontal line crossing landmark 1 was drawn perpendicular to the vertical line. The white (or red) spot size was defined as the sum of the total area of the white (or red) spots on a hind wing divided by the total hind-wing area (the area bounded by a dotted line in Fig. 3A) measured in ImageJ 18 image analysis software. In the majority of specimens (N = 347), the white spot was distinct from the background black area and from red areas, and was easily identified in the image. However, in some specimens (N = 3 before 1993, N = 18 after 1993), such distinction was unclear, because adjoining white and red spots were partially fused. In such cases, we chose 30 points from the clearly white spot area at random with the 'multi-point' tool of ImageJ, converted the image to grey scale, and evaluated the brightness value (0-255) of each pixel. We defined a pixel as being included in the white spot area when its brightness value was higher than the mean − SD brightness value of the focal butterfly. When this procedure was tested on the specimens with distinct white spots, the number of pixels judged as white spot area was almost exactly the same as that identified visually. We first analysed red and white spot sizes separately, and then analysed their genetic correlation.
We similarly measured the spot sizes of the model, P. aristolochiae, for comparison.  Estimation of heritability of mimicry traits. Adult females (N = 16) of P. polytes f. polytes were collected in Nakijin village, Okinawa Island, in 2011. Offspring of those females were reared in the laboratory (25 ± 1 °C, L/D 12:12) on fresh Toddalia asiatica leaves for three consecutive generations. Breeding was random on the condition that mated pairs shared neither parents nor grandparents in order to avoid inbreeding. Each adult was allowed to mate only once. Because the mimicry is a female-limited trait, we used grandmother-granddaughter regression to estimate the heritability of spot size, separately analysing paternal grandmother-granddaughter (for estimation of narrow-sense h 2 ) and maternal grandmother-granddaughter (for estimation of broad-sense H 2 ) regressions 19 . To estimate heritability from the phenotypic similarity between second-degree relatives, the correlation coefficient among grandmother-granddaughter pairs, r (with a confidence interval estimated by bootstrapping), is quadrupled: h 2 (or H 2 ) = 4r 19 . The 95% confidence intervals (CIs) of the heritabilities were estimated by the bootstrap method, using 10 000 repetitions for each parameter set. Genetic correlation between white and red spot sizes is also estimated similarly to the above, except for the correlation was that between grandmother red (white) spot and granddaughter white (red) spot.
Change in spot size over time. We searched in museums and private collections for specimens of the mimetic form of P. polytes collected during the last 55 years (1961-2016) on Okinawa, and in 2011-2016 collected specimens ourselves as well ( Table 2). After phenotypic measurements, we analysed changes in the mimetic trait over time. To compare the trends before and after the new model's arrival, we performed least squares linear regression analysis for 1961-1991 (before the new model's arrival) and 1994-2016 (after). We also searched museum and private collections for the mimetic form captured on other islands in the Ryukyus, namely the Yaeyama group (where the model arrived in 1968) and the Miyako group (where it arrived in 1975) 17 . The small sample size before the 1980s hindered comparison before and after the model's settlement. So we combined the data from the Yaeyamas and the Miyakos for linear regressions of white spot size against year, using data sets before and after 1993. We predicted that white spot size has also changed on those islands but at a different time from that on Okinawa Island.

Statistical analyses.
We used R v. 3.2.3 software 20 for all statistical analyses. We first tested the fit of the white and red spot sizes to a normal distribution by the one-sample Kolmogorov-Smirnov test (in the ks.test package), separately testing the field-collected samples (temporal change analysis for the samples from Okinawa and other islands separately) and the laboratory-reared samples (heritability estimation). In all samples, neither distribution deviated significantly from normal. In all subsequent analyses we used general linear models assuming a normal distribution. We calculated the correlation of spot size between granddaughters and grandmothers for the estimation of heritability and CI as explained above. We performed a least squares linear regression of white spot size against year to detect any change over time. The significance of correlation and regression coefficients was tested using F statistics. Variance in white spot size on Okinawa between before and after arrival was compared by F-test.