A well-known family of butterflies have circular patterns on their wings that look like eyes. These eye-like markings help deflect predators away from the butterfly’s body so they attack the outer edges of their wings. However, in certain seasons, such as the dry season in Africa, the best way for this family to survive is by not drawing any attention to their bodies. Thus, butterflies born during this season shrink the size of their eyespots so they can hide among the dry leaves.

How this family of butterflies are able to change the size of these eye-like spots has only been studied in the species Bicyclus anynana. During development low temperatures, which signify the beginning of the dry season, reduce the amount of a hormone called 20E circulating in the blood of this species. This changes the behavior of hormone-sensitive cells in the eyespots making them smaller in size. But it remains unclear how B. anynana evolved this remarkable tactic and whether its relatives have similar abilities.

Now, Bhardwaj et al. show that B. anynana is the only one of its relatives that can amend the size of its eyespots in response to temperature changes. In the experiments, 13 different species of butterflies, mostly from the family that has eyespots, were developed under two different temperatures. Low temperatures caused 20E hormone levels to decrease in all 13 species. However, most of these species did not develop smaller eyespots in response to this temperature change. This includes species that are known to have larger and smaller eyespots depending on the season.

Like B. anynana, four of the species studied have receptors for the 20E hormone at the center of their eyespots. However, changing 20E hormone levels in these species did not reduce eyespot size.

These results show that although temperature changes alter hormone levels in a number of species, only B. anynana have taken advantage of this mechanism to regulate eyespot size. In addition, Bhardwaj et al. found that this unique mechanism evolved from several genetic changes over millions of years. Other species likely use other environmental cues to trigger seasonal changes in the size of their eyespots.

There are two disparate views regarding phenotypic plasticity. One regards plasticity as a derived adaptation to help organisms survive in variable environments (Bradshaw, 1965; De Jong, 2005) while the other views plasticity as the outcome of flexible, non-canalized, developmental processes, ancestrally present in most organisms, that helps them colonize or adapt to novel environments, a type of pre-adaptation (Newman and Müller, 2000; West-Eberhard, 2003; Pigliucci, 2005; Laland et al., 2014). While both views of plasticity are likely valid, they both currently lack a rigorous, mechanistic examination of ancestral and derived states and direction of change (Bradshaw, 1965). Furthermore, the two views on phenotypic plasticity articulated above, as an adaptation or a pre-adaptation, require either that plasticity evolves under natural selection or that it is ancestral and widespread and facilitates adaptation. Several case studies have been documented in support of the first (Moran, 1992; Wund et al., 2008; Standen et al., 2014) and second evolutionary scenarios (Kiontke and Fitch, 2010; Hiyama et al., 2012) but to date, almost nothing is known about how the plastic responses underlying both scenarios originated and evolved at the proximate, mechanistic level. Details of how plasticity evolves, and whether or not it is widespread and ancestral to a group of species, regardless of their current living environments, may also help discriminate between plasticity being a facilitator or a consequence of organismal adaptation.

Here we focus our investigation on the mechanistic evolution of an adaptive seasonal polyphenism where environmental cues experienced during development alter adult phenotypes to make them fit different seasonal recurrent environments, a highly evolved form of phenotypic plasticity. We use dramatic seasonal variation in the size of B. anynana wing eyespot patterns as our case study. Bicyclus species live throughout dry and wet seasons in Africa, where eyespots of different sizes serve different ecological roles (Brakefield and Reitsma, 1991; Brakefield et al., 1996). In the hot wet season, the large exposed ventral eyespots help deflect attacks of invertebrate predators, or naïve vertebrate predators towards the wing margins (Lyytinen et al., 2004; Olofsson et al., 2010; Prudic et al., 2015), whereas in the cool dry season the smaller eyespots help in camouflage against vertebrate predation (Lyytinen et al., 2004).

Because eyespot size plasticity in B. anynana is sufficiently well understood at the molecular level, this species becomes an ideal springboard for a comparative approach that addresses the mechanistic evolution of this form of plasticity across a phylogeny. Eyespot size plasticity in B. anynana is mostly controlled by temperature, which leads to variable titers of the hormone 20-hydroxyecdysone (20E) at the wandering (Wr) stage of larval development (Monteiro et al., 2015). Manipulations of 20E signaling alone, at that time in development, are sufficient to modify eyespot size (Monteiro et al., 2015). This is because these central cells express the 20E receptor, Ecdysone Receptor (EcR), and upon sufficient 20E signaling, the active 20E-EcR complex is able to interact with yet unknown downstream genes to make these central cells divide and produce a larger central group of signaling cells (Bhardwaj et al., 2017) and ultimately a larger eyespot. Given knowledge of how eyespot size plasticity functions in this species, we sought to investigate how this system of temperature sensitivity evolved by performing a comparative study across nymphalid butterflies, with and without eyespots.

Eyespots originated once within the nymphalid family, about 85 mya, likely from pre-existing simple spots of a single color (Oliver et al., 2012; Oliver et al., 2014) but it is unclear whether size plasticity in response to temperature evolved before or after the origin of eyespots. If eyespot or spot size plasticity is an ancestral pre-adaptation, it is possible that even species of butterflies that do not experience seasonal environments (such as those living near the equator), might have the ability to develop different eyespot or spot sizes when reared at different temperatures under experimental conditions. Alternatively, if eyespot size plasticity is an evolved adaptation, used exclusively by species living in seasonal environments, then only these species should exhibit plasticity.

To test these hypotheses and to examine how plasticity in B. anynana evolved, we reared twelve species from different nymphalid sub-families, and from tropical, or sub-tropical regions, plus one outgroup papilionid species (Figure 1—source data 1—Table S1) at two different temperatures, separated by 10°C, and measured spot and eyespot size plasticity in adult females. Three different types of reaction norm to rearing temperature were observed across species (Figure 1A). Five species showed no significant difference in hindwing (HW) Cu1 spot/eyespot size when reared across two temperatures and were deemed not plastic. Most species showed a decrease in spot/eyespot size with an increase in temperature and had a negative slope in their reaction norms. B. anynana was the only species which displayed a positive slope in its reaction norm, where eyespot size increased with temperature (Figure 1—source data—Table S2). Ancestral character state reconstructions for the slope of these reaction norms suggested that eyespot size plasticity of any form is a derived trait within nymphalids, with three possible independent origins. Ancestral species of nymphalids lacked plasticity, whereas there were one or two independent origins of a negative response of eyespot size to increasing temperature and a separate origin of the opposing pattern of plasticity in ventral HW eyespot size in the Satyrid lineage, such as those leading to B. anynana (Figure 1B).

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Figure 1—source data 1

Supporting details for Figure 1.

Table S1 - Species reared for comparative morphometrics, gene expression and hormonal measurements. Table S2 - F statistics, p-values from analysis of covariance for differences in Cu1 eyespot size between rearing temperatures (fixed factor) and assigned character state for phylogenetic analysis.

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To investigate the molecular basis for how these distinct patterns of plasticity differed from that of B. anynana we compared 20E titers and EcR expression across species using female data, and focused exclusively on examining data for the critical period of development that was previously discovered for the regulation of Cu1 eyespot size plasticity in ventral hindwings of B. anynana, for example the wandering stage of larval development. 20E titers at the wandering stage were consistently higher at the higher rearing temperature across all butterflies (Figure 2A) (Figure 2—source data 1), suggesting that 20E titer plasticity in response to temperature is an ancestral trait shared across these butterflies. EcR expression at the wandering stage was absent in species with no central wing spots (e.g., Danaus), it was absent from spot centers in species with simple spots (e.g., Papilio and Idea), but was present in the eyespot central cells across all other species investigated, with a few exceptions (Junonia coenia (Koch et al., 2003) and Junonia almana) (Figure 2B). Species with no EcR staining in spots still had EcR expression in the large polyploid nuclei that make up the peripodial membrane that wraps around each larval wing (Figure 2—figure supplement 1). In contrast, the spot and eyespot focal marker gene Spalt, was present in all species with eyespots or with simpler spots, as previously reported (Oliver et al., 2012; Stoehr et al., 2013). The absence of EcR expression in J coenia eyespots is restricted to the wandering stage of development, as EcR is expressed in the eyespots of this species in later stages of development (Koch et al., 2003). The EcR expression data overall suggests that EcR localization in eyespots, at the wandering stages of development, is a derived trait, present only in species with eyespots. Some of these eyespotted species subsequently lost EcR expression at this stage of development, a phenomenon previously reported for forewing Cu1 ventral eyespots in B. anynana females (Monteiro et al., 2015).

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F statistics, p-values from analysis of covariance for differences in 20E hormone titers between rearing temperatures (fixed factor) and assigned character states for phylogenetic analysis.

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Finally, to test whether eyespots expressing EcR are size-regulated by 20E we manipulated 20E levels and EcR-mediated signaling directly, focusing again, exclusively on the wandering stages of development, previously shown to be the critical hormone-sensitive stage in B. anynana. Functional experiments were performed in four species of butterflies from different Nymphalid subfamilies, Idea leuconoe (Danainae), a control outgroup danainae with no EcR expression in its black spots, Vindula dejone (Nymphalinae), Doleschallia bisaltide (Nymphalinae), and B. anynana (Satyrinae), the latter three displaying EcR expression in their eyespot centers. Our prediction would be that Idea should not respond to 20E signaling at all, given the lack of the receptor in its spots, and that increases in 20E signaling at low temperature might cause the eyespots of Vindula and Doleschalia to become smaller but those of B. anynana to become larger, whereas decreases of 20E signaling at high temperature might cause the eyespots of the first two species to become larger but smaller in B. anynana. Injections of 20E into female wanderers reared at low temperature (and with lower 20E titers) and of an EcR antagonist, CucB, into female wanderers reared at high temperature (and with higher 20E titers), showed no response across the first three species, whereas eyespot size significantly increased with 20E injections and decreased with antagonist injections in B. anynana (Figure 3). These data indicate that only the eyespots of B. anynana are sensitive to 20E signaling at the wandering stage, within the natural range of titers displayed by these species. This sensitivity is a derived trait potentially restricted to the satyrid sub-family within nymphalids (Figure 4).

Figure 3

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Mean body weight of wandering larvae, hemolymph volume and natural 20E titers at two different rearing temperatures; 20E and CucB injection volume.

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While multiple reports have focused on the role of hormones as mediators of developmental plasticity in a variety of traits (Emlen and Nijhout, 1999; Dai et al., 2001; Gotoh et al., 2011; Gotoh et al., 2014), the physiological and developmental details of how a fully functional plastic trait evolves during the course of evolution were still obscure. Here, we identified the approximate evolutionary origins of individual components of a plastic response of eyespot size in response to temperature and discovered this plastic response to be a complex trait that evolved gradually via changes to different molecular components. Our work showed that the evolution of plasticity in hormone titers, the evolution of hormone receptor expression in the trait, and the evolution of eyespot sensitivity to these hormones all took place at different stages of nymphalid diversification (Figure 4).

An increase in eyespot size in response to temperature appears to be restricted to satyrid butterflies, and is a derived response within nymphalids. Plasticity in eyespot size in butterflies had been primarily documented in satyrid butterflies such as Melanitis leda and several Bicyclus species (Brakefield, 1987; Roskam and Brakefield, 1999; van Bergen et al., 2017) where size was always found to increase with rearing temperature. Most of the reared species of nymphalids and the papilionid species showed a slight decrease in eyespot/spot size with an increase in temperature, while some species showed no plasticity at all. This decrease in eyespot size with increasing temperature may simply reflect non-adaptive variation from a poorly canalized system. In addition, satyrid butterflies, but none of the other species, used the 20E asymmetry to regulate the size of their eyespots in a novel way. This was enabled by the prior recruitment of EcR to the eyespot central cells perhaps concurrently with eyespot origins (Figure 4). These central signaling cells play an important role in determining eyespot size at the wandering stages of development (Monteiro et al., 1994). Some species, such as Junonia coenia, retain expression of EcR in eyespots but only at other stages of wing development (Koch et al., 2003). Finally, the active 20E-EcR complexes increase eyespot size in B. anynana but not in other species with similar EcR expression in their eyespot centers. The ability of 20E to promote localized patterns of cell division might have evolved in the lineage leading to B. anynana alone (Bhardwaj et al., 2017).

Eyespot size plasticity in connection with wet and dry seasonal forms is widely conserved across the sub-family Nymphalinae (Clarke, 2017) but our results suggest that different mechanisms may have evolved to regulate eyespot size plasticity in these lineages. Our controlled rearing experiments showed that all nymphalinae (Vanessa cardui, Junonia almana, J. coenia, J. atlites, J. iphita and Doleschallia bisaltide) produced only small changes in the size of the their Cu1 eyespots in response to rearing temperature, and these were in the opposite direction to those observed in B. anynana. Other environmental factors might cue and regulate these species' seasonal morphs (Figure 1—figure supplement 1), perhaps cues that better predict the arrival of the seasons where these butterflies have evolved. Investigations at the proximate level will be required to correctly establish the environmental cues that induce seasonal forms in these other butterfly species. In addition, a broader temporal investigation of the critical developmental periods of environmental sensitivity, followed by investigations of hormonal sensitivity at those critical periods, will need to be performed for these other species in future. Future investigations can also expand the evolution of plasticity to eyespots not examined here. For now, we uncover phenotypic plasticity in ventral (Cu1) eyespot size in B. anynana hindwings as a complex, step-wise adaptation to seasonal environments cued by temperature that required very specific mutations to evolve. This work also serves as a warning that if many forms of adaptive plasticity are as specific and hard to evolve as the one documented in B. anynana, these exquisite adaptations to specific predictable fluctuating environments may in fact, lend the species vulnerable to extinction under unpredictable climate change, as previously noted (Oostra et al., 2018).

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1-4.

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Author details

1. Shivam Bhardwaj

   Department of Biological Sciences, National University of Singapore, Singapore, Singapore

   Present address

   Department of Biological Sciences, Mississippi State University, Starkville, USA

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   For correspondence

   sb@u.nus.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-5030-7826

2. Lim Si-Hui Jolander

   Department of Biochemistry, National University of Singapore, Singapore, Singapore

   Contribution

   Formal analysis, Investigation

   Competing interests

   No competing interests declared

3. Markus R Wenk

   1. Department of Biological Sciences, National University of Singapore, Singapore, Singapore
   2. Department of Biochemistry, National University of Singapore, Singapore, Singapore

   Contribution

   Investigation, Project administration

   Competing interests

   No competing interests declared

4. Jeffrey C Oliver

   Office of Digital Innovation & Stewardship, University of Arizona, Tucson, United States

   Contribution

   Software, Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2160-1086

5. H Frederik Nijhout

   Department of Biology, Duke University, Durham, United States

   Contribution

   Resources, Investigation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-5436-5345

6. Antonia Monteiro

   1. Department of Biological Sciences, National University of Singapore, Singapore, Singapore
   2. Yale-NUS College, Singapore, Singapore

   Contribution

   Conceptualization, Supervision, Funding acquisition, Project administration, Writing—review and editing

   For correspondence

   antonia.monteiro@nus.edu.sg

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-9696-459X

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