Phenotypic plasticity of wing color patterns revealed by temperature and chemical applications in a nymphalid butterfly Vanessa indica

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Journal of Thermal Biology 33 (2008) 128–139 www.elsevier.com/locate/jtherbio

Phenotypic plasticity of wing color patterns revealed by temperature and chemical applications in a nymphalid butterfly Vanessa indica Joji M. Otaki Laboratory of Cell and Functional Biology, Department of Chemistry, Biology and Marine Science, Faculty of Science, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0213, Japan Received 11 May 2007; accepted 23 November 2007

Abstract 1. A physiological approach was taken to reveal a spectrum of phenotypic plasticity of butterfly wing color patterns using a nymphalid butterfly Vanessa indica. 2. A long-term low-temperature treatment or heat-shock treatment produced various modification types, characterized by the expanded or reduced black spots on the proximal forewing, the reduced white band on the dorsal forewing, and unique hindwing patterns. Ecdysteroid injection produced individuals with paler coloration overall. 3. Variation of these modified color patterns was phenotypically similar to the natural phenotypic variation of species in the genus Vanessa, whose implications were discussed in the light of an evolutionary role of phenotypic plasticity. r 2007 Elsevier Ltd. All rights reserved. Keywords: Phenotypic plasticity; Color-pattern modification; Temperature treatment; Ecdysteroid; Vanessa indica

1. Introduction The color-pattern formation of butterfly wings has been receiving much attention as an experimental system to study a genetic basis for animal development and evolution, partly due to the fact that the butterfly wing color patterns are tantalizing example of how organisms express diverse phenotypes in the course of development and evolution (Beldade and Brakefield, 2002; McMillan et al., 2002; Beldade et al., 2005, 2006). The butterfly wing is a two-dimensional developmental system where each immature scale cell determines its identity based on positional information, which provides us with a technical opportunity to perform simple experimental manipulations together with molecular biological methods (Nijhout, 1985, 1991; Brakefield and French, 1995; French and Brakefield, 1995; Brakefield et al., 1996; Monteiro et al., 2006). Butterfly biology is historically independent of but strongly influenced by developmental and evolutionary studies of Tel./fax: +81 98 895 8557.

E-mail address: [email protected] 0306-4565/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jtherbio.2007.11.004

the insect model system, the fruit fly Drosophila melanogaster (Lawrence, 1992; Carroll et al., 2001). However, a conventional genetic approach to the butterfly wing system, methodologically similar to the Drosophila genetics, is not always straightforward. To artificially make a series of genetic mutants whose aberrant phenotypes are highly specific to the wing color patterns has not been very successful. Probably because many developmental genes are used both in embryogenesis and in adult development, mutations specific to the wing color patterns may be embryonic lethal. More practically, it is not a trivial matter to keep lepidopteran insects for many generations to employ them in genetic experiments with the exception of a few established lepidopteran models such as the domesticated silkworm moth Bombyx mori. One of the solutions to this problem is the ongoing genetic and genomic analysis of the African satyrine butterfly Bicyclus anynana, which may pave a way to the advancement of butterfly biology (Beldade and Brakefield, 2003; Beldade et al., 2005, 2006). An alternative approach is to make aberrant phenotypes by physiological means such as temperature and chemical treatments (Otaki, 1998), which is a technical and

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conceptual application of the pioneering studies by Nijhout (1984) and Shapiro (1984). This approach is successful in making wing color-pattern-specific ‘‘phenocopies’’, which could suggest, at least theoretically, the existence of their corresponding genetic mutants. A few unique modification types induced by temperature and chemical treatments were already produced (Otaki, 1998, 2003, 2007a, b; Otaki and Yamamoto, 2003, 2004b; Umebachi and Osanai, 2003; Otaki et al., 2005; Serfas and Carroll, 2005). The induced modifications suggested a possible mechanism of the colorpattern determination, although its molecular basis is a focus of future research (Otaki and Yamamoto, 2004a; Otaki et al., 2006a, b). It is important, from the viewpoint of developmental biology, to reveal not only a single or few modification type(s) but also a spectrum of plastic phenotypes of a given species, which would suggest more detailed mechanism of the color-pattern determination. Equally important, the physiological approach is more than an alternative to the genetic approach. Because it is phenotype, rather than genotype, that is the direct target of natural selection, phenotypic plasticity of an ancestral species under a given environment could determine the direction of divergent selection in the course of speciation (Scheiner, 1993; Rundle and Nosil, 2005; Pigliucci et al., 2006; West-Eberhard, 1989, 2005). It is highly informative to reveal phenotypic plasticity of a given species through physiological means to infer its evolutionary roles (Waddington, 1953, 1956). For example, the induced modifications of lycaenid butterflies seem to be related to the phenotypic diversity of this family (Otaki and Yamamoto, 2003; Otaki and Kudo, unpublished data). Likewise, the induced modifications of the Indian Red Admiral butterfly Vanessa indica appear to be related to the natural phenotypic diversity in the genus Vanessa sensu stricto (Otaki and Yamamoto, 2004a; Otaki et al., 2006a, b). That is, we have already pointed out that the natural phenotypic variation of the genus Vanessa can be quantified at least in part by the relative area of orange (RAO) on the forewing and that the similar variation can be obtained experimentally by the physiological induction of color-pattern modifications in V. indica. However, the phenotypic variation of the genus Vanessa cannot be explained solely by the variation of the orange area, and other modification types are expected to be identified. As an experimental treatment to obtain modified color patterns, cold-shock application has been widely employed in several species of butterflies. For example, Nijhout (1984) employed 2 1C for 24 or 72 h immediately after pupation using Vanessa cardui, Vanessa virginiensis, and Junonia coenia, and successfully produced a linear series of the cold-shock-induced phenotype. The present study focuses on the developmental and evolutionary aspects of phenotypic plasticity using V. indica, which is commonly seen in the Ryukyu Archipelago, Japan. To reveal a spectrum of phenotypic plasticity for the wing color patterns, here I employed a long-term low-temperature treatment that was milder than the conventional cold-

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shock treatment as well as heat-shock and chemical treatments. We know that V. indica is distributed widely from tropical to frigid regions, although it is essentially a temperate species (Field, 1971; Tsukada, 1985; Otaki et al., 2006a, b; Vane-Wright and Hughes, 2007). For example, Sakhalin, the suspected northern range margin of this species in the Eastern Asia (Asahi et al., 1999), has a long low-temperature period from September to May. Thus, near the northern range margin including Sakhalin, it is highly likely that V. indica occasionally experiences such a relatively long low-temperature period in the field. It is also likely to experience high temperature in tropical areas in southern Asia (Tsukada, 1985). On the other hand, treatment with thapsigargin, a plant-derived stressinducing chemical, is considered to be a mimic of environmental stress. Similar defense chemicals may be ingested by larvae from their food plants in the field. These treatments, which mimic relatively rare but naturally occurring conditions, produced several unique modification types that have not been documented yet. Furthermore, simply because ecdysteroid effect on the color-pattern development has been described (Koch and Bu¨ckmann, 1987; Koch, 1995; Rountree and Nijhout, 1995; Koch et al., 1996, 2003; Otaki et al., 2005), 20-hydroxyecdysone was also used for treatment. Together with other information, I discuss their developmental implications in the color-pattern determination and evolutionary implications in the phylogenetic and phenotypic diversity of the genus Vanessa sensu stricto. 2. Materials and methods 2.1. Animals The India Red Admiral butterfly Vanessa indica (Herbst, 1794) was used. Field (1971) was referred to as a standard reference for species that constitute the genus Vanessa sensu lato. For species that are distributed in the Southeast Asian islands, Tsukada (1985) was also referred to. The genus Vanessa sensu stricto consists of nine species at this point including Vanesa abyssinica, whose phylogenetic position is to be solidified in the future (Nakanishi, 1989; Wahlberg et al., 2005; Otaki et al., 2006a, b; Vane-Wright and Hughes, 2007). Eggs were collected from the field-caught females, or larvae were collected from the field in Ishigaki-jima Island and Okinawa-jima Island, both of which constitute the Ryukyu Archipelago, Japan. Okinawa-jima Island was located southwest of the Mainland Japan, and Ishigakijima island further southwest, close to Taiwan. Larvae were fed on the natural host plant Boehmeria nivea and kept at 2772 1C. When these pupae were kept under the same conditions until eclosion, the normal summer form adults were always obtained. Thus, all experiments were performed on pupae that were supposed to become the normal summer form adults in terms of the color pattern. Sex difference was ignored.

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2.2. Experimental treatments For low-temperature treatments, pupae reared at 2772 1C were transferred to an incubator set at 471 1C within 6 h after pupation, and they were kept there for different durations from 2 to 20 days. These treatments were called ‘‘low-temperature’’ treatments (and not cold shock), since the employed temperature was higher than the conventional cold-shock treatments (Nijhout, 1984). For the heat-shock treatment, pupae were transferred to an incubator set at 3871, 3971, or 4071 1C for 1 day (24 h) within 6 h after pupation. Experimental procedures for the systemic injections of pharmacological agents into pupae were described elsewhere (Otaki, 1998, 2007a; Otaki and Yamamoto, 2004a; Otaki et al., 2005). Sodium tungstate, thapsigargin, and 20-hydroxyecdysone were obtained from Sigma-Aldrich (St. Louis, USA). Thapsigargin and 20-hydroxyecdysone were dissolved in dimethyl sulfoxide (DMSO; SigmaAldrich), and these preparations (10 mM, 1 mL for thapsigargin and 1.0 mM, 2 mL for 20-hydroxyecdysone) were injected into pupae that pupated within 6 h. Thapsigargin is a plant-derived cell-permeable intracellular calcium releaser that specifically inhibits calcium-ATPase of endoplasmic reticulum (ER), resulting in the inhibition of protein synthesis or ‘‘ER stress’’ (Treiman et al., 1998; Futami et al., 2005). DMSO itself does not cause any change in the color pattern (Otaki et al., 2005). 2.3. Color pattern and coloration analyses For the color-pattern analysis of each pattern element, we used the terminology of Nijhout (1991) and Scott (1986) together with our conventional terms (Fig. 1). Most elements or traits were expressed as two- or three-letter

codes. To judge whether normal or modified, I simply compared their color patterns with my collection of fieldcaught individuals and also to standard pictorial records: Field (1971), Tsukada (1985), Asahi et al. (1999), and Shiroˆzu (2006). An individual that has an unusual trait never seen in any of these records was considered as ‘‘modified’’. Most modifications were quantitative difference of patterns, but a single trait, orange hue on the ventral hindwing, was completely new and qualitatively different from the normal color patterns (i.e., the trait itself was not seen in the normal individual at all). This trait is abbreviated as OH in Fig. 2. Modification phenotype of an individual was symbolically expressed as (X1, X2), X3, where X1 indicates the character of the black spots on the proximal forewing, X2 the character of the white spots on the distal forewing, and X3 the character of the ventral hindwing (Fig. 1). The modified individuals induced by sodium tungstate were described as (R, R), W (R for reduced and W for tungstateinduced or white) (Fig. 1). Each treated individual by 20-hydroxyecdysone was scored for the modification degree (MD), which was set from MD0 (no modification) to MD3 (most extreme modification) based on the visual inspection on paleness of the overall wing coloration. To quantitatively evaluate these modifications, the following factors were devised: RAO and relative width of white (RWW). The RAO indicates the orange area relative to the whole wing area on the dorsal forewing as defined in Otaki and Yamamoto (2004a) (Fig. 4A). Digital images of wings were obtained through a high-resolution image scanner, CanoScanD1250U2F (Canon, Tokyo), so that the image-taking conditions were always consistent. Using the scanned image of a dorsal forewing, the RAO was calculated as the number of pixels of the orange area divided by the number of pixels of the whole wing, and

Fig. 1. Terminology for color-pattern elements or traits and wing regions of the normal (A) and modified (B) V. indica individuals. Most elements or traits were expressed as two- or three-letter codes. For an experimentally treated individual, only modified elements or traits were indicated by arrows and letter codes (B). The modification type was visually examined for each wing or wing region, i.e., the proximal forewing (for the black spots in the orange background), the distal forewing (for the white spots in the black background), and the ventral hindwing (for the mossy bands, border ocelli, parafocal elements, and marginal white line). The modification type was then expressed as (X1, X2), X3, where X1 indicates the character of the black spots in the proximal forewing, X2 the character of the white spots in the distal forewing, and X3 the character of the ventral hindwing. The normal individuals were designated as (N, N), N (summer form) or (N, N), N0 (winter form, see Fig. 3A) (A) and the tungstate-treated individuals as (R, R), W, where R indicates reduction of the black or white spots and W indicates the tungstate-induced or white-enhanced hindwing pattern (B).

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Fig. 2. Color-pattern modifications induced by low-temperature treatment for 2–15 days (A) and for 20 days (B). (A) The 2-day treatment produced the completely normal individuals. The 10-day treatment induced mild modifications, characterized by the orange-enhanced hindwing pattern (O type). The 12- and 15-day treatments produced a different modification type, characterized by the simplified hindwing pattern (S type). All individuals showed reduction of the black spots on the proximal forewing (R). (B) The 20-day treatment produced various modification types. Modifications obtained by the 12- and 15-day treatments were enhanced, characterized by more reduction of the black spots on the proximal forewing (R type) and more simplified hindwing pattern (S type) together with the reduced white bands on the distal forewing (R type) (left). In addition, the orange-enhanced hindwing pattern (O type) was also produced, but this time with the expanded black spots on the proximal forewing (E type).

expressed as percentage (Fig. 4A inset). For the RWW, the length of the black area along the costal margin from the apex to the wing median location (designated as ‘‘a’’ in Fig. 4B inset) and the width of the white band (WB) (designated as ‘‘b’’ in Fig. 4B inset) were measured by a digital caliper. The RWW was calculated as b/a, and expressed as percentage. Both RAO and RAW were then expressed as mean7SD for each treatment mode. Two-sided unpaired t-test was performed using JSTAT version 10 (2006). In addition, color brightness was employed to express modifications induced by the ecdysteroid treatment. From

digital images of wings that were obtained through a scanner mentioned above, RGB values and their corresponding brightness values (ranging from 0 to 255) of the central part of the orange area in the cell (the major wing compartment) on the dorsal forewing were measured using Adobe Photoshop Elements (Fig. 6B inset). A single individual image was measured five times and their mean values were used for further analysis. The digital brightness values for a species or treatment mode were expressed as mean7SD. The unpaired two-sided t-test was performed between the normal and ecdysteroid-treated V. indica using JSTAT version 10 (2006).

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Efficiency of the induced global color-pattern modifications was quantitatively evaluated based on the following factors: survival rate (SR) and induction rate (IR) as defined in Otaki (1998, 2007a, b) and Otaki and Yamamoto (2004a). The SR was calculated as the number of treated individuals with successful eclosion divided by the total number of treated individuals, and expressed as percentage. The IR was calculated as the number of modified individuals divided by the number of treated individuals with successful eclosion. These two factors for each treatment mode performed in this study were summarized in Table 1. 3. Results 3.1. Low-temperature treatments revealed a spectrum of phenotypic plasticity Durations of the pupal exposure to low temperature were set from 2 to 20 days (n ¼ 40 in total). The exposure for 2 days (n ¼ 5) and 3 days (n ¼ 3) showed no appreciable color-pattern modifications (Fig. 2A for a 2-day individual). They were symbolically described simply as normal individuals (N, N), N (N for normal). In contrast, the exposure for 10 days (n ¼ 5) showed small but appreciable modifications in all the treated individuals (Fig. 2A). In these individuals, the black spots on the proximal forewings were reduced in size and the white bands on the distal forewings were also reduced in size, which were phenotypically very similar to the tungstatetreated modifications. Notably, however, their ventral forewings were modified uniquely with the emergence of the orange area along the costal margin and enhanced white scales. These individuals were described as (R, R), O (R for reduced and O for orange). Individuals exposed for 12 days (n ¼ 4) and 15 days (n ¼ 7) showed yet different color-pattern modifications

(Fig. 2A). The black spots on the proximal forewing and the white spots on the distal forewing did not change clearly. Interestingly, the ventral hindwing was uniquely simplified due to the disappearance or reduced contrast of pattern elements. These individuals were described as (N, N), S (S for simplified). The exposure to low temperature for 20 days (n ¼ 16) produced largely two opposite types of modifications (Fig. 2B). One type, described as (R, R), S, was fundamentally similar to the individuals obtained by the 12- or 15-day exposure with clear reduction of both black and white spots on the forewing and with more simplified pattern on the ventral hindwing. Clear dislocation of the parafocal elements was also observed simultaneously in one individual, which was designated as (R, R), S+W. In contrast, the other type, described as (E, R), O, was similar to the individuals obtained by the 10-day exposure with more enhanced orange and white scales on the ventral hindwing, but in these individuals, the black spots were expanded, not reduced, in size. 3.2. Heat-shock and thapsigargin treatments produced darker color patterns In a different nymphalid species, Junonia almana, heat shock acts antagonistically to cold shock, in which colorpattern elements are dislocated away from the focal elements, a reversed type from the cold-shock-induced and tungstate-induced modifications (Otaki, 2007a). Thus, it was probable that the heat-shock treatment of V. indica may also produce a similar modification type. Since the previous heat-shock study on J. almana was carried out at 38–44 1C (Otaki, 2007a, b), I first tested the heat-shock treatment at 38 1C (n=10). This treatment resulted in three dead individuals (SR=70%) and produced modified individuals with the blacker or darker ventral hindwing (Fig. 3B). This obtained modified

Table 1 Color-pattern modifications of V. indica under different conditions Treatment mode

n

Pupal death or failed eclosion

Modified

SR (%)

IR (%)

Low temperature (LT) 4 1C 2 days Low temperature (LT) 4 1C 3 days Low temperature (LT) 4 1C 10 days Low temperature (LT) 4 1C 12 days Low temperature (LT) 4 1C 15 days Low temperature (LT) 4 1C 20 days Heat shock (HS) 38 1C, 1 day Heat shock (HS) 39 1C, 1 days Heat shock (HS) 40 1C, 3 days Thapsigargin (TG) 10 mM, 1 mL Ecdysteroid (ES) 1.0 mM, 2 mL

5 3 5 4 7 16 10 9 5 10 57

0 0 0 0 1 2 3 7 5 2 25

0 0 5 3 6 14 7 2 0 4 24*

100 100 100 100 86 88 70 22 0 80 56

0 0 100 75 100 100 100 100 Na 100 75

Note: Treatment modes are shown in two-letter codes as follows: LT: low temperature; HS: heat shock; TG: thapsigargin; ES: 20-hydroxyecdysone. Each number in the columns of ‘‘n’’, ‘‘Pupal death or failed eclosion’’, and ‘‘Modified’’ indicates the number of individuals applicable to that category. Abbreviations: n: the number of treated individuals; SR: survival rate (%); IR: induction rate (%); Na: not applicable. * Ecdysteroid-treated modified individuals were scored as MD1 (n ¼ 6), MD2 (n ¼ 11), or MD3 (n ¼ 7).

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Fig. 3. Natural color-pattern variation (A) and color-pattern modifications induced by heat shock (B), and thapsigargin injection (C). (A) In the winter season, the hindwing pattern of V. indica appears to become darker in Okinawa (right, a field-caught individual at the end of February 2007), and it was designated as N0 type. This can be compared with B type (B) and F type (C) and also with S type and O type (Fig. 2). (B) Heat-shock treatments at 38 1C produced both reduced and enhanced black spots on the proximal forewings. In both individuals, the hindwing mossy band was simplified (S type), although the dislocation of the parafocal elements was not observed (left and middle). Heat shock at 39 1C produced individuals covered with black scales more extensively, and this was documented as B type. Although not clear, the parafocal elements seemed to be dislocated away from the focal elements. (C) Thapsigargin injection produced the hindwing pattern whose elemental boundary was fussy, designated as F type.

hindwing phenotype was designated as B type (B for black). The overall coloration of the heat-shocked individuals at 38 1C was darker than that of the low-temperatureinduced phenotypes and the normal summer form and as dark as the natural winter form (N, N), N0 (Fig. 3A) in all wing surfaces. Interestingly, the identical temperature conditions produced phenotypically opposite individuals with expansion or reduction of the forewing black spots in size. These individuals were symbolically described as (E, R), B and (R, R), B. The 39 1C treatment (n ¼ 9) resulted in two live individuals with almost entirely black wings (Fig. 3B) and seven dead individuals (SR ¼ 22%), indicating the severity of this treatment to V. indica. The 40 1C treatment (n ¼ 5) did not produce any live adults (SR ¼ 0%). The hindwing pattern B type was somewhat similar to the S type obtained by the low-temperature treatment, but the parafocal elements might be dislocated away from the focal elements, although not very clear (Fig. 3B). This B type was also different from that of the normal winter form, N0 type (Fig. 3A). Less severe B type had conspicuous white scales, which was somewhat similar to the tungstate-induced W type (Fig. 1B). In addition, the elemental boundaries were obscure in the B type, which was shared with the thapsigargin-injected one (see below, Fig. 3C). Similar, but not identical, modification type was obtained by the injection of a stress-inducing chemical, thapsigargin (n ¼ 10). Survived individuals showed blacker hindwing pattern with fuzzy elemental boundaries, desig-

nated as F type (Fig. 3C), and only minor modifications were seen on the forewing. 3.3. Quantitative evaluation of the modification patterns The modifications on the dorsal forewings were quantified in terms of RAO and RWW, and each treatment mode was compared (Fig. 4). Although visual inspection of specimens indicated their phenotypic difference as discussed above (Figs. 2 and 3), statistically significant RAO difference with p-value less than 0.01 was not obtained for any treatment modes in comparison with the 2-day treatment mode (Fig. 4A). This is partly because in each treatment mode there were individuals that did not show any modifications at all. When these individuals were excluded, much lower p-value less than 0.001 was indeed obtained from the 20-day low-temperature mode (p ¼ 0.0007, degrees of freedom ¼ 9, Student t-test, t-value ¼ 5.04). Heat-shock treatment at 39 1C showed very small RAO values as seen in the blackened wings (Fig. 3), although the number of obtained individuals was just two. Another quantitative descriptor was RWW (Fig. 4B). It showed the statistically significant difference between the 2-day and 10-day low-temperature treatment modes at the level of po0.01 (p ¼ 0.0042, degrees of freedom ¼ 8, Student t-test, t-value ¼ 3.95). Between the 2-day and 38 1C-heatshock treatment modes, more significant difference at the level of po0.001 was obtained (p ¼ 0.0005, degrees of freedom ¼ 10, Student t-test, t-value ¼ 5.09). Although the

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Fig. 4. Quantitiative evaluation of temperature and chemical treatments on the color-pattern modifications. Two factors, RAO (relative area of orange) and RWW (relative width of the white band), were measured, and their mean7SD values are shown in each treatment mode. LT, lowtemperature treatment; HS, heat-shock treatment; TG, thapsigargin treatment. Numbers before LT indicate days of exposure, and numbers after HS indicate treatment temperature. Asterisks are shown for small p-values (*po0.01; **po0.001; ***po0.0001) in comparison with the 2day LT mode. The number of examined individuals was indicated at the bottom of each bar. (A) The RAO values appear to decrease in response to the low-temperature treatment and heat-shock treatment. This quantitative result reflects the expansion of the black spots and black area on the proximal dorsal forewings, (E, X), X phenotype, in the treated individuals. However, such effect was obscured by the existence of non-modified individuals among the treated. Exclusion of such individuals from the 20day LT treatment mode produced small p-value (‘‘20LT Modified’’). Inset indicates how to define the RAO values. (B) The RWW values also appear to decrease in response to treatments. This factor is more variable than the RAO, which was especially seen in the 10- and 20-day LT treatment modes with large error bars. This variability is probably dependent on genetic background. Exclusion of non-modified individuals from the 20-day LT treatment mode produced very small p-value (‘‘20LT Modified’’). Inset indicates how to define the RWW values.

20-day low-temperature treatment mode did not show any statistically significant difference from the 2-day mode, this was because some individuals were not affected by the 20day treatment at all. Exclusion of such non-affected individuals readily showed the highly significant difference

Fig. 5. Ecdysteroid injection experiment. (A) Ecdysteroid injection produced paler coloration overall without clear modification of pattern elements themselves. Nevertheless, the hindwing pattern was designated as ES type to note a clearly different phenotype from the N type. Based on the visual inspection of the overall coloration, modification degrees from MD1 to MD3 were assigned to each modified individuals. (B) Brightness of the orange area of seven species that belong to the genus Vanessa sensu stricto and ecdysteroid-treated MD3 individuals of V. indica. The inset with an arrow indicates the orange area where the brightness values were measured. Each color of a bar represents the mean RGB color for each species or the ES-MD3 individuals of V. indica. The number of examined individuals was indicated at the bottom of each bar.

from the 2-day mode, po0.0001 (degrees of freedom ¼ 12, Student t-test, t-value ¼ 8.24). 3.4. 20-Hydroxyecdysone induced the overall coloration change Ecdysteroids have been described to induce the colorpattern change in a few species of nymphalid butterflies

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(Koch and Bu¨ckmann, 1987; Koch, 1995; Rountree and Nijhout, 1995; Koch et al., 1996, 2003; Otaki et al., 2005). To test a modification property of ecdysteroids, 20hydroxyecdysone was injected into pupae (n ¼ 57), resulting in 25 dead individuals and 32 adult individuals with successful eclosion (SR ¼ 56%). Among the survived, 24 individuals showed appreciable change of the overall coloration (i.e., low contrast and paler coloration) on all wing surfaces, although the element-specific modifications such as dislocations, reduction, and enlargement were not observed (Fig. 5A). Their hindwings showed very different color contrast from the normal one, and for this reason, these individuals were designated as (N, N), ES (ES for ecdysteroid). Degrees of the paleness varied among individuals, and these modified individuals were ranked from MD1 to MD3, where MD3 was the palest. In contrast, the induced paler coloration in the orange area was reminiscent of other species of Vanessa such as V. dejeanii and V. dilecta. To further validate this visual inspection, the orange coloration degrees of each species of Vanessa sensu stricto were quantitatively evaluated and compared, which may reflect the developmental and evolutionary ecdysteroid status in each species (Fig. 5B). As expected from the visual inspection, each species had specific color and brightness of its own, from the darkest species, V. atalanta, to the palest species, V. dejeanii. The coloration value of the ecdysteroid-treated individuals of V. indica was comparable to that of V. dilecta or V. dejeanii. Statistical difference at the level of p ¼ 0.0021 (degrees of freedom ¼ 14, Student t-test, t-value ¼ 3.76) was obtained between the normal V. indica individuals and the ecdysteroid-treated MD3 individuals. 4. Discussion 4.1. Developmental implications of phenotypic plasticity This study revealed a spectrum of phenotypic plasticity of the Indian Red Adminal butterfly, V. indica. Whereas the conventional cold-shock and other related treatments were reported to produce a single modification type (Nijhout, 1984; Shapiro, 1984; Otaki, 1998; Otaki and Yamamoto, 2004b; Serfas and Carroll, 2005), the longterm low-temperature conditions employed in this study produced several different phenotypes that have not been known before. Under such conditions, the developmental physiological system may be disturbed, and the system seems to stochastically respond to this disturbance, resulting in the haphazard modification types. Alternatively, but not mutually exclusively, the variable-induced phenotypes may reflect a genetic background that is different from individual to individual. Under the disturbed conditions, both R and E phenotypes of the proximal forewings were obtained, which may reflect the fluctuation of the putative cold-shock hormone (CSH) or its receptors, as suggested by the previous studies (Otaki, 1998; Otaki and Yamamoto, 2004b). Somewhat

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surprisingly, the proximal and distal forewing surfaces responded independently. That is, for the proximal forewing R type, the distal forewing N, E, and R types were all appeared. Furthermore, both S- and O-type individuals of the hindwings were obtained under the identical conditions. In one individual, the S type was expressed together with the influence of CSH, W type, as seen from the dislocations of the parafocal elements, and it was designated as S+W. In contrast, the O type possibly reflects the influence of activities of pigment-synthesizing enzymes in scale cells. These results illustrate an accidental aspect of developmental process of fate determination or developmental noise. Phenotypic variation of the ventral hindwings appears to be larger than that of the forewing. As modified phenotypes, four types were identified in this paper excluding ES type: O, S, B, and F types (Fig. 6). However, I admit that the distinction among S, B, and F types are not very clear; they are all dark phenotypes and one could easily find intermediate individuals among them if more individuals were subjected to treatments. Thus, at this point, it is reasonable to think that the darker phenotypes were produced from a similar mechanism as a result of general stress response (Otaki et al., 2005; Otaki, 2007b) (Fig. 6). These variable phenotypes obtained under the long-term low-temperature exposure were certainly not numerous but limited in number (Table 2). Although there could be more phenotypes to be revealed by different treatments, this fact is consistent with the interpretation that only a few physiological variables are likely to be responsible for the global color-pattern determination process. Accordingly, as a hypothesis, this study proposes that the wing-wide global color-pattern modulators are CSH (which causes W type), ecdysteroids (which causes ES type), and the enzymatic activities in scale cells (which causes O type or dark types). In different nymphalid butterflies, J. coenia and J. almana, injections of dextran sulfate and other related chemicals induced the partially reversed phenotype, in which only the peripheral elements were dislocated away from the focal elements (Serfas and Carroll, 2005; Otaki, 2007a). I speculated that the sensitivity of the peripheral elements but not the focal elements to these chemicals originated from the molecular difference of the expressed CSH receptors. If so, in contrast to such modification type, pharmacological modifications of the focal elements only without affecting the peripheral elements can be expected, although not obtained yet. An interesting point of this study was the observation that the response profile was different in different wing surfaces and pattern elements. The most sensitive element to a treatment was the metallic blue element located on the distal ventral forewing. This element was almost always affected by any treatment, although its deformed shape was variable from individual to individual. Likewise, the white band on the distal forewing shows relatively consistent reduction response in any individual in any treatment, as

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Fig. 6. Summary of the ventral hindwing color patterns in normal and modified individuals. The hindwing color patterns are designated as N or N0 for the normal individuals. Different treatments produced differently modified color patterns. The whole hindwing and its characteristic part are shown as a pair to clarify the modified part.

Table 2 Experimentally induced wing color-pattern phenotypes of V. indica Proximal forewings: Distal forewings: Hindwings:

R, E R W, O, S, B, F, ES

Abbreviations: R, reduced; E, expanded or enhanced; W, white-enhanced; O, orange-enhanced; S, simplified; B, black-enhanced; F, fussy; ES, ecdysteroid treated.

shown by the RWW values (Fig. 4B). In contrast, the black spots on the proximal forewing were reduced in an individual and expanded in another individual even in response to the identical conditions, which made it difficult to statistically show quantitative RAO difference between treatment modes. The color patterns of the ventral hindwings were even more variable, as seen in the six types summarized in Fig. 6 and Table 2. Mechanistic reasons for these response differences are to be explained in the future. It is important to point out here that the positional relations among pattern elements were not changed by the low-temperature treatments and the injection of thapsigargin and 20-hydroxyecdysone. This is in sharp contrast to the tungstate and cold-shock treatments, which may act on the CSH pathway, modifying the interpretation of positional information in scale cells (Otaki, 1998). On the other hand, ecdysteroids, and to some extent, thapsigargin,

probably change the enzymatic activities for the pigment synthesis in scale cells (Koch, 1995; Koch et al., 1996, 2003). At least in J. almana, the ecdysteroid and CSH pathways could cross-talk with each other (Otaki, 2007a), adding further complexity for the wing color patterns. 4.2. Evolutionary implications of phenotypic plasticity It has already been shown that the RAO value of V. indica can be changed by the tungstate treatment, which is reminiscent of the RAO variation of the normal species that constitute the genus Vanessa sensu stricto (Otaki and Yamamoto, 2004a). At the same time, it indicates the importance of environmental thermal conditions for the butterfly color-pattern development and evolution. This fact is consistent with the hypothesis that the CSH or its signal transduction pathway is exploited in the course of speciation of Vanessa (Otaki and Yamamoto, 2004a). In addition to the variation of the black or orange area, other phenotypic variations among Vanessa species including the complexity and brightness of the ventral hindwing pattern and the different overall wing coloration were also to be explained from the viewpoint of phenotypic plasticity and evolution. The quantitative evaluation of brightness of the orange area indicated that each species has a specific brightness value and that the ecdysteroid-injected V. indica certainly expressed paler orange coloration, being closer to V. dilecta and V. dejeanii (Fig. 5). These observations

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support the notion that the ecdysteroid secretion or its signal transduction pathway was changed in the course of the color-pattern evolution and speciation in this genus. It is worthwhile to point out that the ecdysteroid control of the seasonal polyphenism of wing color patterns has been demonstrated in other butterflies (Koch and Bu¨ckmann, 1987; Koch, 1995; Rountree and Nijhout, 1995; Koch et al., 1996). Moreover, the low-temperature treatments revealed the unique hindwing modifications with the orange-based novel trait (O type) or with darker coloration (S, B, and F types). This variation of modified patterns is reminiscent of that of natural Vanessa species (Fig. 7). More specifically, the orange hindwing pattern O type is reminiscent of that of V. samani and V. tameamea (and to some extent, V. dejeanii). The black and fussy patterns B type and F type are reminiscent of the pattern of V. atalanta (and to some extent, V. dejeanii), which does not have much white outlines for each elements but has the

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darker coloration overall on the ventral hindwing. The pattern of the ecdysteroid-treated individuals, ES type, is reminiscent of that of V. tameamea and V. samani. In addition to the direct influence of hormonal factors, the cell-autonomous activities of cellular pigment synthesizing enzymes might have been changed in the course of evolution, which is probably the case in the ventral hindwing, because it exhibited various phenotypes upon treatments. Interestingly, different wing surfaces or wing parts appeared to employ somewhat different evolutionary mechanisms in the course of speciation. These results should be discussed in the light of a proposed evolutionary role of phenotypic plasticity (Scheiner, 1993; Rundle and Nosil, 2005; Pigliucci et al., 2006; WestEberhard, 1989, 2005). Since the natural color-pattern variation of the genus Vanessa sensu stricto roughly corresponds to that of the experimental modification types of V. indica, I here propose a plausible evolutionary history of this genus. If V. indica still retains a spectrum of

Fig. 7. Comparison of the hindwing phenotypes among the treated individuals of V. indica (A) and among the normal Vanessa species (B). The CuA1 cells of the ventral hindwings are shown. The normal phenotypes can be divided into three groups: the normal type (V. indica, V. buana, and V. dilecta), the dark/simplified type (V. atalanta and V. dejeanii), and the orange type (V. samani and V. tameamea). This categorization nicely corresponds to the RAObased one, i.e., the intermediate type, the black type, and the orange type, as indicated at the bottom of this figure. Treatment modes are shown in twoletter codes as follows: ST: sodium tungstate; HS: heat shock; TG: thapsigargin; LT: low temperature; ES: 20-hydroxyecdysone.

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phenotypic plasticity that was held in the ancestral species of Vanessa (Otaki et al., 2006a, b), the Vanessa genus might have exploited the phenotypic plasticity of their ancestor to increase or decrease the orange area and also to change other phenotypic features in the course of the color-pattern evolution and speciation. The coincidence of the plastic phenotypes and the natural phenotypic variation could further support the hypothesis that the possible evolutionary direction was already pre-determined upon the emergence of the ancestral Vanessa species within a spectrum of phenotypic plasticity. Natural selection then enhanced one of the opportunities of plasticity, and it was assimilated in a population during speciation. In summary, the ancestral species of Vanessa already had inherent genetic and physiological plasticity that could potentially be expressed in response to different environmental conditions. No other phenotype beyond this plasticity was allowed. Small populations of this ancestral species were isolated under different environmental conditions. Natural selection then amplified a specific phenotype of plasticity, depending on the environmental conditions. In this evolutionary history of Vanessa speciation, random mutation does not play any role. This could be a typical process of allopatric speciation, although its generality is unknown at this point. 4.3. Possible genetic assimilation of a plastic phenotype in other butterflies One of the aberrant forms of V. indica caught in the field is similar, if not identical, to the cold-shock or tungstatetreated individuals (Otaki and Yamamoto, 2004b), but no field-caught aberrant forms being similar to the new experimental modification types reported in this paper can be found in a collection of Japanese lepidopterological journals as long as I know, indicating the extreme rareness of these modification types in the field at least in Japan. This is in contrast to the cold-shock-induced phenotypes, which were occasionally reported (Otaki and Yamamoto, 2004b). It is somewhat surprising to find that such extremely rare phenotypes can be experimentally produced relatively easily in the present day V. indica. Nonetheless, in its range margins, V. indica would have certainly been exposed to the natural conditions that are similar to the experimental ones performed in this paper. It is not uncommon to find cases where field-caught aberrant individuals (and hence their corresponding types of experimental phenocopies) show clear resemblance to other related species. In lycaenid butterflies, the expansion and reduction of black spots seen in the experimental treatments (Otaki and Yamamoto, 2004b; Otaki and Kudo, unpublished data) show high degrees of resemblance to other species (Otaki and Yamamoto, 2003). It seems that the contrasting color patterns of two Japanese Maculinea butterflies were fixed in the course of evolution in response to the environmental cold shock (Otaki and Yamamoto, 2003). In pierid butterflies, a field-caught

aberrant form and a tungstate-induced modification type of Colias erate resembled Colias palaeno, a closely related species living in the high altitude in Japan (Otaki and Yamamoto, 2004b). In papilionid butterflies, an experimentally induced melanic aberrant form of Papilio xuthus (Umebachi and Osanai, 2003) and a field-caught melanic form of Papilio machaon (Nishiyama, 2001), a possible temperature-induced phenotype, showed a striking similarity to the natural form of the melanic P. machaon in North America. Indeed, the P. machaon group is known to show various width of the yellow and black bands on the wings (Nijhout, 1991; Sperling, 2003). These cases may be explained as evolutionary exploitation of phenotypic plasticity. If so, in all the four butterfly families within Papilionoidea, Papilionidae, Pieridae, Lycaenidae, and Nymphalidae (de Jong et al., 1996; Ackery et al., 1999), plastic color patterns with respect to environmental thermal conditions are likely to have been exploited during the color-pattern evolution and speciation. Acknowledgments This work was partly supported by the Sumitomo Foundations Environmental Research Grant and also by the 21st century COE program of the University of the Ryukyus from the Ministry of Education, Culture, Sports, Science and Technology, Japan. References Ackery, P.R., de Jong, R., Vane-Wright, R.I., 1999. The butterflies: Hedyloidea, Hesperioidea and Papilionoidea. In: Kristensen, N.P. (Ed.), Lepidoptera, Moths and Butterflies. 1. Evolution, Systematics and Biogeography. Handbook of Zoology 4 (35), Lepidoptera. de Gruyter, Berlin, pp. 263–300. Asahi, J., Kanda, S., Kawata, M., Kohara, Y., 1999. The Butterflies of Sakhalin in Nature. Hokkaido Shinbun-sha, Sapporo, Japan. Beldade, P., Brakefield, P.M., 2002. The genetics and evo-devo of butterfly wing patterns. Nat. Rev. Genet. 3, 442–452. Beldade, P., Brakefield, P.M., 2003. Concerted evolution and developmental integration in modular butterfly wing patterns. Evol. Dev. 5, 169–179. Beldade, P., Brakefield, P.M., Long, A.D., 2005. Generating phenotypic variation: prospects from ‘‘evo-devo’’ research on Bicyclus anynana wing patterns. Evol. Dev. 7, 101–107. Beldade, P., Rudd, S., Gruber, J.D., Long, A.D., 2006. A wing expressed sequence tag resource for Bicyclus anynana butterflies, an evo-devo model. BMC Genomics 7, 130. Brakefield, P.M., French, V., 1995. Eyespot development on butterfly wings: the epidermal response to damage. Dev. Biol. 168, 98–111. Brakefield, P.M., Gates, J., Keys, D., Kesbeke, F., Wijngaarden, P.J., Monteiro, A., French, V., Carroll, S.B., 1996. Development, plasticity and evolution of butterfly eyespot patterns. Nature 384, 236–242. Carroll, S.B., Grenier, J.K., Weatherbee, S.D., 2001. From DNA to Diversity: Molecular Genetics and the Evolution of Animal Design. Blackwell Science, Malden. de Jong, R., Vane-Wright, R.I., Ackery, P.R., 1996. The higher classification of butterflies (Lepidoptera): problems and prospects. Entomol. Scand. 27, 65–101. Field, W.D., 1971. Butterflies of the Genus Vanessa and of the Resurrected Genera Bassaris and Cynthia (Lepidoptera: Nymphalidae). Smithsonian

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