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. 2020 Feb 11;9:e49544. doi: 10.7554/eLife.49544

Origin of the mechanism of phenotypic plasticity in satyrid butterfly eyespots

Shivam Bhardwaj 1,†,✉, Lim Si-Hui Jolander 2, Markus R Wenk 1,2, Jeffrey C Oliver 3, H Frederik Nijhout 4, Antonia Monteiro 1,5,✉
Editors: Patricia J Wittkopp6, Karen E Sears7
PMCID: PMC7012602  PMID: 32041684

Abstract

Plasticity is often regarded as a derived adaptation to help organisms survive in variable but predictable environments, however, we currently lack a rigorous, mechanistic examination of how plasticity evolves in a large comparative framework. Here, we show that phenotypic plasticity in eyespot size in response to environmental temperature observed in Bicyclus anynana satyrid butterflies is a complex derived adaptation of this lineage. By reconstructing the evolution of known physiological and molecular components of eyespot size plasticity in a comparative framework, we showed that 20E titer plasticity in response to temperature is a pre-adaptation shared by all butterfly species examined, whereas expression of EcR in eyespot centers, and eyespot sensitivity to 20E, are both derived traits found only in a subset of species with eyespots.

Research organism: Other

eLife digest

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.

Introduction

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.

Results

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).

Figure 1. Eyespot/spot size plasticity is widespread across butterfly lineages but the response to rearing temperature has different norms of reaction across species.

(A) Size of hindwing ventral Cu1 eyespots (arrowheads). Thirteen species of butterflies were reared at two different rearing temperatures. Eyespot size corrected for wing size is plotted for two different temperatures (low temperature 17°C or 20°C is marked with blue symbols, while high temperature of 27°C or 30°C is marked with red symbols). Error bars represent 95% CI of means. (B) Mapping origins of eyespot size plasticity via maximum parsimony phylogenetic analysis suggests three independent origins for two different patterns of plasticity in the lineage with eyespots (eyespot size decreases with increasing temperatures: red lineages, and eyespot size increases with increasing temperature: blue lineage). The lineage leading to Satyrid butterflies gained a positive response to plasticity (blue arrowhead), whereas most other Nymphalids had either no response, or limited negative plasticity response (red arrowhead).

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.

Figure 1.

Figure 1—figure supplement 1. Phenotypic plasticity in wing patterns is observed across a wide variety of species in wild.

Figure 1—figure supplement 1.

Figure 1—figure supplement 2. Data from Wahlberg et al. (2009) show high support value for the basal nodes across different analysis methods.

Figure 1—figure supplement 2.

Figure 1—figure supplement 3. Ancestral state reconstruction using maximum likelihood models suggest that positive directionality of plasticity is a derived trait in Satyrid butterflies.

Figure 1—figure supplement 3.

Ancestral states are significantly positive (blue outline), significantly negative (red outline), or not significantly different from zero (gray outline).

Figure 1—figure supplement 4. Tree used for ancestral state hypotheses tests.

Figure 1—figure supplement 4.

See text and Figure 4—source data 1 for explanation of node numbers.
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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).

Figure 2. 20E titers increase with rearing temperature across most species but EcR expression is only found in a subset of nymphalids with eyespots.

(A) 20E titers increase with an increase in rearing temperature across most species. This trait is ancestral in nature, with a likely origin before the origin of eyespots. (B) EcR is absent in simple spots, but present in the future eyespot centers of most of the species investigated (N ≥ 3 for each immunostaining: numbers in superscript represent sample size; Scale bars,10µm).

Figure 2—source data 1. 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.

Figure 2.

Figure 2—figure supplement 1. EcR expression as a positive control in peripodial membrane nuclei acrossspecies.

Figure 2—figure supplement 1.

(N ≥ 3 for each species, Scale bars, 10µm).

Figure 2—figure supplement 2. 20E titers increase with increasing temperatures as an ancestral trait, present at the basal node of Nymphalids and outgroups.

Figure 2—figure supplement 2.

All ancestral states are not significantly different from zero (gray outline).
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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. Sensitivity of eyespots to EcR-mediated signaling evolved in the lineage leading to B. anynana butterflies.

Figure 3.

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Four species of butterflies were injected with 20E hormones or EcR antagonists (CucB) during the wandering (Wr) stage. Control larvae were injected with an equal volume solution of saline vehicle (V). While Idea leuconoe, Vindula dejone and Doleschallia bisaltide are not sensitive to either of the hormone signal manipulations, B. anynana shows sensitivity towards both 20E and CucB. Error bars represent 95% CI of means. Significant differences between treatments are represented by asterisks: **, p<0.01, ***, p<0.001.

Figure 3—source data 1. Mean body weight of wandering larvae, hemolymph volume and natural 20E titers at two different rearing temperatures; 20E and CucB injection volume.

Figure 4. Phenotypic plasticity as a complex trait evolved gradually.

Figure 4.

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Phylogenetic analysis suggests three independent origins for two different patterns of eyespot size plasticity (eyespot size decreases with increasing temperatures: red lineages and red circles, and eyespot size increases with increasing temperature: blue lineage and blue circles). Empty circles represent a lack of plastic response. Green circles (character state 1) represent high 20E titers with increasing temperature, while white circles (character state 0) represent no significant difference in titers at two developmental temperatures. Green squares represent presence of EcR in eyespots, while white squares represent its absence. EcR expression in eyespots is inferred to have originated concurrently with the origin of eyespots, about 85 Mya, and subsequently lost in a few nymphalid lineages. Green triangles represent sensitivity towards 20E (character state 1), while white triangles represent absence of sensitivity (character state 0). Question marks represent missing data points. Circles, square and triangle on left with vertical bars represent respective estimated evolution of eyespot size plasticity (red and blue circles), 20E titer plasticity (green circle), EcR expression in eyespots (green square) and sensitivity towards 20E (green triangle). Alternative models using Maximum Likelihood reach similar conclusions (Supplementary Information: Figure 1—figure supplement 2, 3, Figure 4—source data 1). H. iboina image copyright of David.C. Lees, Cambridge University Department of Zoology.

Figure 4—source data 1. Results of likelihood ratio tests and AIC comparisons.
See Figure 1—figure supplement 1 for node identities.

Discussion

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).

Materials and methods

Key resources table.

Reagent type
(species) or resource		 Designation Source or reference Identifiers Additional
information
Strain, strain background (Butterflies, females) Junonia atlites; Junonia coenia; Junonia iphita;
Junonia almana; Doleschallia bisaltide; Vanessa cardui; Vindula dejone; Cethosia cynae; Bicyclus anynana; Morpho peleides; Danaus chryssipus; Idea leuconoe;
Papilio polytes 		Penang Butterfly Farm, Malaysia;
Duke University;
Yale University;
National University of Singapore
Antibody EcR common isoform, Manduca sexta DSHB 1:10
Antibody Spalt, Primary antibody, Guinea pig Stoehr et al., 2013 1:20000
Antibody AlexaFlour 488 green Goat anti-mouse secondary antibody Molecular Probes Cat# A-11001, RRID:AB_2534069 1:800
Antibody Goat anti-Guinea pig secondary antibody Molecular Probes Cat# A-11076, RRID:AB_141930 1:800
Chemical compound, drug 20-Hydroxyecdysone (20E) Sigma-Aldrich Cat# H5142 Lot # 060M1390V
Chemical compound, drug Cucurbitacin B (CucB) Sigma–Aldrich Cat# C8499 Lot # 035M47104V
Software, algorithm Imaris v8.64 (ImarisXT, Bitplane AG)
Software, algorithm Rphylopars (Goolsby et al., 2017)
Software, algorithm ape (Paradis et al., 2004)
Software, algorithm packages for R (R Development Core Team, 2018)
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Butterfly husbandry

All species were reared at two temperatures separated by 10 degrees, at 70 or 80% RH and at 12:12 hr light: dark cycle. The only exception was Junonia coenia, which was reared at 16:8 hr light: dark cycle and 80% RH. B. anynana was reared in climate control chambers in Singapore, at 17°C and 27°C, at 80% RH. Vanessa cardui, and Morpho peleides were reared in climate control chambers at Yale University, New Haven at 17°C and 27°C, and at 80% RH. Junonia coenia was reared at 20° and 30°C at Duke University. All other species of butterflies were reared at Entopia (formerly, Penang Butterfly Farm, Penang, Malaysia) in temperature-controlled chambers (PT2499 Incubator, Exoreptiles, Malaysia) at 20°C and 30°C, and at 70% RH. Humidity in these latter chambers was monitored using (PT2470 Hygrometer, Exoreptiles, Malaysia) and EL-USB-2 data loggers (Lascar Electronics, PA 16505, USA).

Four hours after emergence, butterflies were captured and frozen in glassine envelopes at −20°C. All larvae in this experiment were sexed during larval or pupal stages and only females were used for analysis. Wings were carefully dissected and imaged using a Leica upright microscope. Wing images were processed in ImageJ, where area and eyespot size were measured using selection tools.

Additional data for Nymphalid species not reared in Figure 1—source data 1—Table S1 but mentioned in Figure 1, Figure 4, Figure 1—figure supplement 2 and Figure 1—figure supplement 3 were extracted and analyzed from van Bergen et al. (2017). These species were reared at 21°C and 27°C, and slopes were corrected for a temperature difference of 6°C, instead of usual 10°C for other species used in this study.

Hemolymph collection

Previous studies in B. anynana have pointed to the wandering (Wr) stage as the critical temperature sensitive stage for determination of ventral hindwing eyespot size (Monteiro et al., 2015). Time lapse photographs of larval development were captured every 15 min using a RICOH camera to determine the beginning of the Wr stage across all species. Initiation of Wr stage is marked by the larvae stopping to feed, purging their gut, and starting to wander away from the food and looking for a place to pupate. Using Hamilton syringes, 20 µL of hemolymph, were extracted from each larvae at ~70% development in Wr stage (15 hr after Wr started for animals reared at 30°C, and 25 hr for animals reared at 20°C). Extracted hemolymph was then dissolved in freshly prepared 90 µl methanol + 90 µl isooctane and stored at −20°C until hormone extraction (Bhardwaj et al., 2017) .

Wing tissue collection

Larval wing discs were dissected from Wr stage larvae at 27°C or 30°C and stored in fix buffer until further processing at 4°C. These were later stained for EcR expression using a primary antibody 15F1 (DSHB) raised against a Manduca sexta EcR peptide shared across all isoforms of EcR. AlexaFlour 488 green Goat anti-mouse (Thermo Fisher Scientific Cat# A-11001, RRID:AB_2534069) was used as secondary antibody at a dilution of 1:800 for EcR stains. Primary antibodies against Spalt, a previously published (Stoehr et al., 2013) nuclear marker for spots and eyespots, was used at a dilution of 1:20000, supported with Goat anti-Guinea pig secondary antibody (Molecular Probes Cat# A-11076, RRID:AB_141930; at a dilution of 1:800), as a location marker for putative eyespots/spots in the larval wings. Serial optical sections of the Cu1 eyespot wing sector were imaged using LSM510 Meta, to distinguish between dorsal and ventral surfaces. Specific slices were obtained from raw images using Imaris v8.64 (ImarisXT, Bitplane AG, software available at http://bitplane.com. Junonia coenia EcR data were taken from Koch et al. (2003).

20E and antagonist injections

Four species of butterflies, Idea leuconoe, Vindula dejone, Doleschallia bisaltide, and B. anynana, were injected with 20E or CucB during the Wr stage. Injections were made at ~50% development of Wr stage (12–14 hr at 30°C, 18–22 hr at 20°C; For B.anynana, rearing were done at 27°C and 17°C, respectively). Average body weights of wandering larvae and total hemolymph present were calculated for each species, and used to calculate naturally circulating 20E levels in vivo. A gradient of different concentrations of 20E and CucB were used for pilot experiments. Maximum concentrations of 20E, which did not surpass the natural levels, and of CucB, which did not cause mortality or pupation defects, were used for injections and are summarized in the table below. 20E and CucB were dissolved in 10% EtOH to make working solution for injections. Equal volume injections of Vehicle (10% EtOH in Saline) injections were done as controls (Figure 3—source data 1). After injections, animals were reared at their regular rearing temperature (17°C for B.anynana, 20°C for other 20E injected animals and 27°C for B.anynana, 30°C for CucB injected animals) until emergence as adults. After emergence, the wings were dissected, imaged, and scored for further analysis.

Statistical analysis

All wing and eyespot data were log10 transformed to ensure linearity of wing size with eyespot size for purposes of allometric scaling and regression analysis, and to be able to compare slopes across species with different eyespot sizes and wing sizes. Univariate ANCOVAs were performed using hindwing Cu1 eyespot area as the main variable, hindwing area as a covariate, and rearing temperature as a fixed factor in SPSS v21. Graphs were plotted in Microsoft Office 2016 for Mac. Slopes for plasticity of eyespot size and 20E titers were measured using the expression:

Slope=(Valueathightemperature−Valueatlowtemperature)Differenceinrearingtemperature(10∘C)

Using reverse transformed data for eyespot size, and untreated values for hormone titers.

Phylogenetic analysis

Patterns of plasticity in eyespot size were categorised in distinct groups based on positive, negative, or slopes undistinguishable from zero when eyespot size was plotted against temperature. Using a pruned version of a larger phylogenetic tree for all nymphalid genera (Wahlberg et al., 2009; Oliver, 2013), ancestral trait reconstructions were performed, and evolution of the reaction norm slopes was mapped using maximum parsimony in Mesquite. Similar analyses were performed using data obtained for hormone titer plasticity where species were categorized into two categories – those with a positive slope or a zero slope, and data for presence or absence of EcR expression, and 20E-EcR signaling affecting eyespot size.

We also evaluated several hypotheses concerning the evolution of relevant traits with likelihood ratio tests (LRT) and Akaike Information Criteria (AIC). For all analyses, specific ancestral nodes of interest were 'fixed' for a particular state and the resultant maximum likelihood score was used for LRT and AIC comparisons (Oliver et al., 2012). We performed four tests in all, investigating (1) whether the most recent common ancestor (MRCA) to all butterflies (node 14 in Figure 1—figure supplement 4) had plasticity in spot and eyespot size or not; (2) whether the MRCA to all butterfly species with eyespots (node 17) had plasticity in eyespot size or not; (3) whether the MRCA to all butterflies (node 14) had positive hormone titre plasticity or not; and (4) whether the MRCA to all butterfly species with eyespots (node 17) expressed EcR in the locations of future spots / eyespots or not. For tests of eyespot size plasticity, we used a three-state coding scheme: positive size plasticity, negative size plasticity, and no plasticity. Character states were scored based on the sign of the slope of the reaction norm; species with reaction norms that were not significantly different from zero were scored as having no temperature-dependent plasticity in eyespot size (Figure 1—source data 1—Table S2). Tests on positive hormone titer plasticty and EcR expression used characters coded as binary states. For AIC comparisons, we used the correction for small sample sizes (AICc) and evaluated models based on the AICc weight, wi = e((min(AICc – AICc)/2). Models were considered significantly different if they differed by 2 or more log-likelihood units or the AICc weight was less than 0.2.

For all comparisons, there was little significant support for one hypothesis over another (Figure 4—source data 1). In tests on the origin of eyespot size plasticity, both the MRCA to all butterflies and the MRCA to all butterflies with eyespots had slightly better likelihood and AICc scores for being non-plastic than being plastic. Positive hormone titer plasticity in the MRCA to all butterflies had more support than a non-plastic MRCA, although the difference in likelihoods and AICc was not significant. Finally, the absence of EcR expression in the MRCA of all eyespot-bearing butterflies had higher likelihood and AICc scores than a model in which the MRCA did express EcR in future spot / eyespot centers. The absence of significant support for one model over another is largely due to the low number of species examined.

Phylogenetic analysis of continuous reaction norms

We also analyzed the continuous-valued measures of four plasticity traits: slope of size plasticity in the ventral hind wing Cu1 eyespot and slopes of titer plasticity in juvenile hormone, ecdysone, and 20E, Using the Rphylopars (Goolsby et al., 2017) and ape (Paradis et al., 2004), packages for R (R Development Core Team, 2018). We estimated ancestral states separately for each trait, using the anc.recon function in Rphylopars. For each ancestral node, we used the 95% confidence interval to determine significance: if zero was excluded from the 95% C.I., the ancestral state was categorized as having a significantly non-zero slope.

Data and materials availability

All data is available in the main text or the supplementary materials.

Acknowledgements

We thank Ms. Mei Lee Wong, Mr. Andy Loke and Mr. BT Chin (Penang Butterfly farm, Malaysia) for their support and supplies of butterflies used in these experiments. We thank Ms. Jocelyn Wee for facilitating confocal imaging. We acknowledge Anne K Bendt for excellent SLING scientific program management and operations support. The EcR 10F1-s developed by Riddiford, LM was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242.

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Contributor Information

Shivam Bhardwaj, Email: sb@u.nus.edu.

Antonia Monteiro, Email: antonia.monteiro@nus.edu.sg.

Patricia J Wittkopp, University of Michigan, United States.

Karen E Sears, University of California, Los Angeles, United States.

Funding Information

This paper was supported by the following grants:

  • Ministry of Education - Singapore MOE2014-T2-1-146 to Antonia Monteiro.

  • National Research Foundation Singapore NRFI2015-05 to Markus R Wenk.

  • National University of Singapore LSI to Markus R Wenk.

  • Agency for Science, Technology and Research BMRC-SERC 112 148 0006 to Markus R Wenk.

  • National University of Singapore NUS Research Scholarship to Shivam Bhardwaj.

Additional information

Competing interests

No competing interests declared.

Author contributions

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

Formal analysis, Investigation.

Investigation, Project administration.

Software, Formal analysis, Investigation, Methodology.

Resources, Investigation.

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

Additional files

Transparent reporting form

Data availability

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.

References

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Decision letter

Editor: Karen E Sears1
Reviewed by: Christopher Wheat

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

Through its investigation of a well-studied trait in a remarkably charismatic and diverse group, this article has the potential to become a textbook example of the evolution of developmental plasticity. The article represents an impressive amount of research, and the approach taken, in which the authors worked backwards from the focal species to determine how the trait evolved was deemed "incredibly thought provoking and insightful" by reviewers. This article will be of benefit to eLife readers with interests in developmental biology, evo-devo, phenotypic plasticity, phenotypic evolution, and insect and butterfly evolution.

Decision letter after peer review:

[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]

Thank you for submitting your work entitled "Origin of phenotypic plasticity in butterfly eyespots" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by a Senior Editor. The reviewers have opted to remain anonymous.

Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.

While all the reviewers appreciate the potential importance of the work completed by the authors, reviewer #3 in particular, a noted expert in this field, notes several fatal flaws in the study's design and the experimental implementation which prevent us from publishing the study in eLife. I strongly suggest that the authors review the comments of reviewer #3 before submitting their research to another journal.

Reviewer #2:

I thoroughly enjoyed reading this manuscript by Bhardwaj and colleagues. This is exciting, novel and truly unique research. The comparison of plastic responses using evo-devo responses across 12 species is unprecedented. The results suggest that the complex hormonal interactions that result in seasonal plasticity in Bicyclus butterflies originate in a step-wise manner – ecdysone sensitivity to temperature predates the plastic response, while ecdysone receptor expression in the eye spot area, and sensitivity to ecdysone are derived in the lineage with seasonal plasticity. These results have important implications for our understanding of the evolution of plasticity, and complex traits more generally. The figures in this manuscript are excellent visualizations of the main findings and patterns of phylogenetic variation. However, several clarifications are necessary with respect to the treatment of plasticity, and incorporation of literature outside of butterflies.

First, the manuscript as currently written is focused primarily on butterflies (e.g., from the second paragraph onwards), but the results are applicable to a much broader range of systems. It would be great if related examples from other systems could be incorporated in the Introduction and Discussion. For instance, work of JW Thornton on estrogen receptor evolution speaks to both regulators of plasticity (e.g., sex differences as developmental plasticity) and step-wise evolution of complex interactions between signaling molecules and receptors (e.g., preadaptation of ligands or receptors, see Thornton et al., 2003 Science, Thornton, 2001 PNAS, Bridgham et al., 2006 Science).

Second, the working definition of plasticity in this manuscript is fairly specific to polyphenisms, and other forms of evolved, developmental switches (sensu MJ West-Eberhard). This is important, because it affects some of the assumptions in the Introduction and the conclusions in the Discussion. For instance, the Introduction states that "we still have little understanding of how these complex adaptive responses originate and evolve across a phylogeny" and the Discussion concludes that "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." These statements seem very much specific to "highly evolved" forms of plasticity like polyphenisms where developmental modules are switched on or off in response to an environmental cue. These statements seem less applicable to other forms of plasticity. For instance, Newman and Muller argued that plasticity – in terms of environmental sensitivity – is the ancestral state for most phenotypes, and that developmental canalization of originally environmentally induced body plans is derived (2000, "Epigenetic mechanisms of character origination"). In another case, consider trial-and-error learning as a type of plasticity – there are a plethora of studies looking at brain size and learning across a phylogeny, and these forms of plasticity are considerably less "fragile" with respect to performance in completely novel conditions. I suggest that the manuscript is framed around polyphenisms specifically, or evolved molecular interactions related to plasticity.

In terms of the methods, I have only one minor concern. I understand the challenges of rearing 12 species of butterflies, especially when they require different host plants and rearing conditions. However, the species were reared in three different locations, and Bicyclus anynana was reared in a unique location. It would be worth discussing in the Materials and methods the possible role of variation in rearing environment – is there any chance the differences across the species could be environmental rather than characteristics of the species?

Reviewer #3:

This manuscript takes a comparative look at temperature-controlled plasticity in the size of butterfly wing spot color patterns ("eyespots"). The study attempts to take a phylogenetic approach to infer the evolutionary history of different aspects of the eyespot temperature response mechanism. The authors look at the effect of rearing temperature on eyespot size across 13 species, which is an interesting and valuable exercise. They also look at temperature effects on titers of a hormone (20E) that affect color pattern polyphenism in some butterfly species, as well as expression of EcR, the presumed 20E receptor. Lastly, they look at the effects of injecting 20E, as well as a receptor antagonist, at one time point in four species. From these comparative datasets they make a phylogenetic argument that eyespot plasticity in one particular species occurred through gradual assembly of a novel hormone response mechanism. My overall thoughts are that the motivation of the work is very interesting, and there are some valuable data here. But there are also numerous fatal flaws in the study, in terms of experimental design and data quality (described below) that cause me to be highly skeptical about the authors' interpretation of the data.

Important conclusions of the paper are based mostly on negative results. My strongest criticism of this entire study, as it is with many other evo-devo papers that foray into phylogenetic character analysis, is that major conclusions are built on poorly controlled negative data. In fact, for almost every trait that is subject to phylogenetic analysis in this paper, I have serious concerns about problems with poor time series sampling, sampling at the wrong developmental stage, drawing homologies between species, etc. And the worry with all of these issues is negative data. Negative results from various species are counted as character states in a phylogenetic analysis which lead the authors to their conclusion about evolution of a developmental mechanism. But, as I describe below, there is strong reason to believe that a lack of time series data (and in one case, apparently wilful ignoring of published data) is biasing this work towards producing negative results.

Photoperiod is known to affect wing pattern plasticity, but was not controlled for between treatments and species. Rearing photoperiod is different between some species, and is simply not reported for many other species (I cannot find the information in the manuscript). This is a seriously confounding factor for the results in Figure 1 since it is known that photoperiod has a strong effect on plastic wing pattern traits in many butterfly species, both independent of, and in interaction with, temperature. Without photoperiod controls one cannot determine how much of a phenotypic response shown in Figure 1 is due to temperature vs. other environmental effects. To be publishable, for all species the photoperiod has to be the same for both temperature treatments, and photoperiod has to be reported in the paper.

There is no evidence that the Papilio, Danaus, and Idea "eyespots" share evolutionary or developmental homology with true nymphalid eyespots. I am unaware of any developmental, gene expression, or genetic data supporting homology of these color patterns with true eyespots. In my opinion these patterns are more likely to be derived from central or margin WntA patterning systems. They look just like patterns that are lost in WntA CRISPR knockouts in other species (Mazo-Vargas et al., 2017). If the authors' homology call is incorrect, then any data gained from analyzing these color patterns will completely distort the phylogenetic analysis. This potential problem is especially critical since these are outgroups.

EcR antibody stains are poor quality, lack proper controls, and are only for a single stage. The EcR immunostains in Figure 2 are important data for assembling the phylogenetic model. Unfortunately there are many problems with these data and they are not publishable as shown. First of all, there are no positive controls for the species that are scored as having no immunofluorescent signal. Do we know that antibody works in these species?

Second, there are no time expression series. Perhaps in the negative cases expression EcR a bit earlier or later. I am very perplexed by this lack of consideration for other time points besides larval wandering stage. For example, EcR expression has been published in Junonia (Precis) coenia eyespots at a later timepoint, in correlation with the eyespot transcription factor Distalless no less (Koch et al., 2003). But the authors completely ignore this in their model, and even score J. coenia EcR expression as negative in their phylogenetic analysis. I think this ignoring of different time points, and previously published data, is surprising and I hope not purposefully misleading. I also find it very odd that there are no J. coenia EcR immunostains in this paper, especially since one of the experts on the species (H. Nijhout) is a co-author on the paper. As I point out on multiple occasions in this review, the authors need to consider more than a single development stage to adequately address their research questions.

Third, some of the stains look poor quality. They don't look like spot expression to me, and the image size, framing, and resolution is poor so I can't tell of the signal is localized to the nucleus as one would expect for EcR. I would like to see a supplemental figure with whole-wing images for all species to see color pattern correlations more clearly and to rule out background fluorescence. I would like to see proper counterstaining. I would like to see positive controls for the EcR antibody in each species. I would like to see higher magnification images showing the purported "spot" patterns, as well as nuclear localizations. If possible, it would also make the data much stronger to see double stains to test for co-localization of EcR and some other eyespot marker. This would be especially important for some of the species in which the stains look more like diffuse smudges in the provided images.

Single-stage sampling for hormone titer comparisons doesn't make sense because critical periods are known to be different for different species. 20E titers only provided for a single timepoint in this study (wandering larvae), because this is the important stage for B. anynana. There are several important problems with this lack of temporal sampling. First of all, across insects the timing of hormone pulses and sensitive periods can be very specific and short-lived, and small changes in these pulses and periods are almost certainly significant for trait evolution. Therefore a single time point, generally, cannot serve as proxy for an entire endocrine process in a cross-species comparative study, at least without some sort of reference time series for each species. Second, this is almost certainly a problem in this study since it is known that the 20E pulse and sensitive period for color pattern polyphenism in J. coenia is at a later time point that is not even sampled in this paper (Rountree and Nijhout, 1995). I am concerned that by sampling only a single timepoint in all the species in this study, there may be important false negatives. Third, I think by not doing time series that there is simply a lot of interesting biology that is being missed. To me it is very interesting that both B. anynana and J. coenia express EcR in their eyespots (the latter observation is mostly ignored in this paper), yet have different sensitive periods and responses. Clearly there is more complex evolution going on here. I would like for this paper to talk more about this. Most importantly, though, they should have titer time series to have a complete, honest, and interesting story.

Staging all injection experiments at a single stage is not sufficient for comparative analysis because it is known that hormone-sensitive periods vary between species. All 20E and CucB injections were at a single stage (the wandering larva). This is problematic because many of the key conclusions of the paper are based on negative results from these injections. For many of the reasons outlined above, including previous experimental data from J. coenia, it is critical to do multiple timepoints. From these single time point injections we cannot rule out the possibility that the non-responsive species may simply be responsive at different time points. I am especially skeptical about V. dejone, which shows EcR expression in the eyespot and has a very strong eyespot temperature response. As I describe elsewhere, I am also skeptical that the black spots in I. leucone are homologous with B. anyana eyespots at all, so I am not sure how meaningful the results from this species are at all.

Odd inconsistency in injection controls. Shouldn't the "V" negative controls be the same between the 20E and CucB experiments? Yet they are extremely different, in some cases requiring completely different axis scales between experiments. In a few cases the differences between the different negative controls is much greater than the differences between the treatment and the negative control. The B. anyana results looks especially strange. Either I am missing something that needs to be explained better, or there was some problem with the experiments.

eLife. 2020 Feb 11;9:e49544. doi: 10.7554/eLife.49544.sa2

Author response

[Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]

Reviewer #2:

I thoroughly enjoyed reading this manuscript by Bhardwaj and colleagues. This is exciting, novel and truly unique research. The comparison of plastic responses using evo-devo responses across 12 species is unprecedented. The results suggest that the complex hormonal interactions that result in seasonal plasticity in Bicyclus butterflies originate in a step-wise manner – ecdysone sensitivity to temperature predates the plastic response, while ecdysone receptor expression in the eye spot area, and sensitivity to ecdysone are derived in the lineage with seasonal plasticity. These results have important implications for our understanding of the evolution of plasticity, and complex traits more generally. The figures in this manuscript are excellent visualizations of the main findings and patterns of phylogenetic variation. However, several clarifications are necessary with respect to the treatment of plasticity, and incorporation of literature outside of butterflies.

First, the manuscript as currently written is focused primarily on butterflies (e.g., from the second paragraph onwards), but the results are applicable to a much broader range of systems. It would be great if related examples from other systems could be incorporated in the Introduction and Discussion. For instance, work of JW Thornton on estrogen receptor evolution speaks to both regulators of plasticity (e.g., sex differences as developmental plasticity) and step-wise evolution of complex interactions between signaling molecules and receptors (e.g., preadaptation of ligands or receptors, see Thornton et al., 2003 Science, Thornton, 2001 PNAS, Bridgham et al., 2006 Science).

Thank you. We have now modified the Introduction to include a more general paragraph about the origin of plasticity across different systems. We have refrained from including the references above (from Thornton et al.) because these references are more concerned with explaining how the steroid receptor family achieved its current diversity, and how receptor ligand systems co-evolve, something that we do not explore in the current work. As far as we know, the steroid receptor EcR is present across all butterfly species examined and in single copy.

Second, the working definition of plasticity in this manuscript is fairly specific to polyphenisms, and other forms of evolved, developmental switches (sensu MJ West-Eberhard). This is important, because it affects some of the assumptions in the Introduction and the conclusions in the Discussion. For instance, the Introduction states that "we still have little understanding of how these complex adaptive responses originate and evolve across a phylogeny" and the Discussion concludes that "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." These statements seem very much specific to "highly evolved" forms of plasticity like polyphenisms where developmental modules are switched on or off in response to an environmental cue. These statements seem less applicable to other forms of plasticity. For instance, Newman and Muller argued that plasticity – in terms of environmental sensitivity – is the ancestral state for most phenotypes, and that developmental canalization of originally environmentally induced body plans is derived (2000, "Epigenetic mechanisms of character origination"). In another case, consider trial-and-error learning as a type of plasticity – there are a plethora of studies looking at brain size and learning across a phylogeny, and these forms of plasticity are considerably less "fragile" with respect to performance in completely novel conditions. I suggest that the manuscript is framed around polyphenisms specifically, or evolved molecular interactions related to plasticity.

Yes, we agree that there are different views on the origins of plasticity and whether or not plasticity is a derived or ancestral state for most organisms. In our opinion both views for the origin of plasticity are actually possible but it is important, in each case, to use the phylogenetic comparative method to explore ancestral states and direction of change, something that is rarely done, especially when examining how plastic systems evolve at the molecular, proximate level. We have included the paragraph below to introduce the distinct views that predominate the field of plasticity followed by explaining that our focus will be on examining the origin of the adaptive plasticity underlying wing pattern polyphenism in satyrids, represented by Bicyclus anynana:

“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 e.g., a pre-adaptation (Newman and Müller, 2000; West-Eberhard, 2003; Pigliucci, 2005; Laland et al., 2014). […] Here we focus our investigation on the mechanistic origins 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.”

In terms of the methods, I have only one minor concern. I understand the challenges of rearing 12 species of butterflies, especially when they require different host plants and rearing conditions. However, the species were reared in three different locations, and Bicyclus anynana was reared in a unique location. It would be worth discussing in the Materials and methods the possible role of variation in rearing environment – is there any chance the differences across the species could be environmental rather than characteristics of the species?

Thank you. We have explained in our methods that these butterflies were reared in climate-controlled chambers across all 3 locations, where we monitored temperature and humidity across larval development. The environmental factors were consistent across locations, which implies that its effects are minimal in this study.

Reviewer #3:

This manuscript takes a comparative look at temperature-controlled plasticity in the size of butterfly wing spot color patterns ("eyespots"). The study attempts to take a phylogenetic approach to infer the evolutionary history of different aspects of the eyespot temperature response mechanism. The authors look at the effect of rearing temperature on eyespot size across 13 species, which is an interesting and valuable exercise. They also look at temperature effects on titers of a hormone (20E) that affect color pattern polyphenism in some butterfly species, as well as expression of EcR, the presumed 20E receptor. Lastly, they look at the effects of injecting 20E, as well as a receptor antagonist, at one time point in four species. From these comparative datasets they make a phylogenetic argument that eyespot plasticity in one particular species occurred through gradual assembly of a novel hormone response mechanism. My overall thoughts are that the motivation of the work is very interesting, and there are some valuable data here. But there are also numerous fatal flaws in the study, in terms of experimental design and data quality (described below) that cause me to be highly skeptical about the authors' interpretation of the data.

Important conclusions of the paper are based mostly on negative results. My strongest criticism of this entire study, as it is with many other evo-devo papers that foray into phylogenetic character analysis, is that major conclusions are built on poorly controlled negative data.

Thank you for your comment. Negative results (supported with appropriate controls) are as informative as positive results. The reviewer assumed that the data was built on poorly controlled negative data but that is not the case. We have included more information on how we controlled for these negative results in the Materials and methods section of the manuscript.

In fact, for almost every trait that is subject to phylogenetic analysis in this paper, I have serious concerns about problems with poor time series sampling, sampling at the wrong developmental stage, drawing homologies between species, etc. And the worry with all of these issues is negative data. Negative results from various species are counted as character states in a phylogenetic analysis which lead the authors to their conclusion about evolution of a developmental mechanism. But, as I describe below, there is strong reason to believe that a lack of time series data (and in one case, apparently willful ignoring of published data) is biasing this work towards producing negative results.

We think this criticism is unfair and misguided. It is also unclear what the reviewer thinks are “negative results”. We did not pursue any particular type of result when setting up to do this study. We merely report what we found. Furthermore, this work represents the most detailed study done to date in Lepidoptera that includes carefully sampled specimens at homologous stages of development. We initially used time-lapse photography to establish the Wr stage as the temperature sensitive stage for ventral Cu1 eyespot size plasticity in a model system (Bicyclus anynana) (Monteiro et al., 2015). Larvae were placed individually in transparent vertical containers with food, and photographed every 15 minutes. When the larvae left the food and climbed the container upwards (without returning) they were marked as having reached the beginning of the wandering stage. We performed the same time-lapse photography across all other species. Quantification of 20E titers, EcR stainings, and 20E injections were all performed based on this careful wandering stage staging to allow us to compare truly homologous points of development across all species. Most of this information was already detailed in the methods in the original submission but we have accentuated this further.

Photoperiod is known to affect wing pattern plasticity, but was not controlled for between treatments and species. Rearing photoperiod is different between some species, and is simply not reported for many other species (I cannot find the information in the manuscript). This is a seriously confounding factor for the results in Figure 1 since it is known that photoperiod has a strong effect on plastic wing pattern traits in many butterfly species, both independent of, and in interaction with, temperature. Without photoperiod controls one cannot determine how much of a phenotypic response shown in Figure 1 is due to temperature vs. other environmental effects. To be publishable, for all species the photoperiod has to be the same for both temperature treatments, and photoperiod has to be reported in the paper.

Thank you for pointing out this important omission. We have now modified the Materials and methods details to include this information:

“All species were reared at two temperatures separated by 10 degrees, at 70 or 80% RH and at 12:12h light: dark cycle. The only exeption was Junonia coenia, which was reared at 16:8h light: dark cycle. […] Humidity in these latter chambers was monitored using a PT2470 Hygrometer (Exoreptiles, Malaysia) and EL-USB-2 data loggers (Lascar Electronics, PA 16505, USA).”

There is no evidence that the Papilio, Danaus, and Idea "eyespots" share evolutionary or developmental homology with true nymphalid eyespots. I am unaware of any developmental, gene expression, or genetic data supporting homology of these color patterns with true eyespots. In my opinion these patterns are more likely to be derived from central or margin WntA patterning systems. They look just like patterns that are lost in WntA CRISPR knockouts in other species (Mazo-Vargas et al., 2017). If the authors' homology call is incorrect, then any data gained from analyzing these color patterns will completely distort the phylogenetic analysis. This potential problem is especially critical since these are outgroups.

Previous work suggested that eyespots originated from existing spots, perhaps like the ones shown in Idea, a Danainae (a basal-branching sub-family of the Nymphalidae) (Oliver et al., 2014). Because of this previous work, we decided to also examine species with simpler spots within and outside the Nymphalidae. The gene Spalt is a known marker for spots and eyespots across Nymphalidae and Pieridae (see Figure S2 of (Oliver et al., 2012), and (Stoehr et al., 2013)) and we have now modified Figure 2 to include Spalt co-stainings performed at the same time as EcR for all species. We observe that spots, localized at homologous positions as Cu1 eyespots, in Papilio and Idea express Spalt during development, just like eyespots. This shared expression pattern suggests (but does not conclusively show) that eyespots and spots may be homologous, and that spots may have preceded eyespots in evolution. Importantly, however, given that all outgroup species with spots did not express EcR in spots or in any region that might be homologous to eyespots, we concluded that the EcR expression pattern is likely to have originated in the sister lineage to the Danainae, which is also the lineage where eyespots originated. In Danaus, another Danainae, the wing margin spots are indeed likely to represent a wing margin patterning system that is impacted by WntA, which may not be homologous to either spots (like in Idea) or eyespots. Regardless, this result, contrary to what the reviewer suggests, does not “completely distort the phylogenetic analysis”. The inclusion of this species has no significant impact on our ancestral state reconstruction for the origin of EcR expression in regions that map to eyespot centers.

EcR antibody stains are poor quality, lack proper controls, and are only for a single stage. The EcR immunostains in Figure 2 are important data for assembling the phylogenetic model. Unfortunately there are many problems with these data and they are not publishable as shown. First of all, there are no positive controls for the species that are scored as having no immunofluorescent signal. Do we know that antibody works in these species?

We have now included a new Supplementary figure (Figure 2—figure supplement 2) to include positive controls of EcR expression in the large nuclei of the peripodial membrane as previously reported for EcR (Koch et al., 2003). In addition, we have added in the main Figure 2 Spalt (Sal) co-stainings because this gene marks future spot and eyespot centres. Sal expression was present across all species examined with spots/eyespots, irrespective of whether they expressed EcR in these pattern elements.

Second, there are no time expression series. Perhaps in the negative cases expression EcR a bit earlier or later. I am very perplexed by this lack of consideration for other time points besides larval wandering stage.

In previous work we established that the wandering stage is the critical sensitive stage of development when ventral Cu1 eyespot size is determined in B. anynana in response to temperature (Monteiro et al.,2015). This was our departure point for this comparative investigation. All animals used in our experiments were monitored during their entire development and we were able to confidently identify the same homologous wandering stage using time lapse photography across all species. Expanding the current work (with 13 species) to include sampling of EcR stainings at other time points would be prohibitive in terms of sampling effort and is not required to answer the question we posed at the beginning of this manuscript. Our work does not preclude future examinations of these alternative time points as candidate developmental stages that regulate plasticity in other species. Note, however, that often different cis-regulatory elements control the expression of the same gene across different body locations and even stages of development at homologous locations. A famous example of the latter are the different cis-elements of the gene Distal-less that initiate expression in early limbs of Drosophila, and then maintain this expression in later stages of limb development (McKay et al., 2009). This implies that the origin of novel cis-elements, or disruption of homologous and pre-existing cis-elements might be responsible for altering the expression pattern of genes over the course of development at homologous positions in the wing over evolutionary time. To avoid these confounding effects we restricted our examination of the molecular evolution of the mechanisms of plasticity in Bicyclus anynana by sampling strictly homologous tissues (ventral wings and Cu1 wing sectors) and time points across a phylogeny.

For example, EcR expression has been published in Junonia (Precis) coenia eyespots at a later timepoint, in correlation with the eyespot transcription factor Distalless no less (Koch et al., 2003). But the authors completely ignore this in their model, and even score J. coenia EcR expression as negative in their phylogenetic analysis. I think this ignoring of different time points, and previously published data, is surprising and I hope not purposefully misleading.

The reviewer is incorrect in assuming that we ignored this previous result. We are well aware of this previous study but for the reasons already outline above, we chose to focus our investigation on strictly homologous time periods of development across all species examined. Relative to J. coenia, previous work documented EcR expression in the peripodial membrane and along the trachea, but not in the focal eyespot central cells at the wandering stage of development (Koch et al., 2003). EcR is later observed in J. coenia eyespots during pupal development, which is a later stage and not the focus of this study. Given that close relatives such as J. atlites and J. iphita have EcR expression in the Cu1 ventral eyespot centers at the wandering stages of development, the most likely evolutionary explanation for the observed absence of EcR in eyespots in this species and also in J. almana, is the loss of this expression pattern at this stage of development. Note that this type of stage-specific EcR regulation has been documented before in B. anynana forewing ventral eyespots (in females only; Monteiro et al., 2015), where EcR is expressed in mid-larval stages and again in early pupal stages, but is absent at the wandering stage – allowing these eyespot centers to be insensitive to temperature. This allows these eyespots to retain the same size in both seasonal forms and function in sexual selection and/or predator avoidance year around. Again, we are neither ignoring other time-points (they are just beyond the scope of current study), nor purposefully misleading the readers.

I also find it very odd that there are no J. coenia EcR immunostains in this paper, especially since one of the experts on the species (H. Nijhout) is a co-author on the paper. As I point out on multiple occasions in this review, the authors need to consider more than a single development stage to adequately address their research questions.

While the reviewer is correct in pointing out that H. Nijhout is an expert and a co-author of the current study, we have carefully reviewed existing literature (performed by Koch and Nijhout) and used their specific EcR expression data from J. coenia (Koch et al., 2003) to conduct our ancestral state reconstructions.

Third, some of the stains look poor quality. They don't look like spot expression to me, and the image size, framing, and resolution is poor so I can't tell of the signal is localized to the nucleus as one would expect for EcR. I would like to see a supplemental figure with whole-wing images for all species to see color pattern correlations more clearly and to rule out background fluorescence. I would like to see proper counterstaining. I would like to see positive controls for the EcR antibody in each species. I would like to see higher magnification images showing the purported "spot" patterns, as well as nuclear localizations. If possible, it would also make the data much stronger to see double stains to test for co-localization of EcR and some other eyespot marker. This would be especially important for some of the species in which the stains look more like diffuse smudges in the provided images.

We have now included double stains for the positive control gene, Spalt, previously shown to mark spots and eyespots, as well as a supporting image for Figure 2, which includes positive controls for EcR stainings in the large nuclei of the peripodial membrane for all species except one (where no peripodial membrane was photographed at the time, and where the samples have since deteriorated). This species, however, had a positive EcR signal in the eyespot centers. The cells of the larval wing disc are very small and punctate nuclear stainings are very difficult to visualize. However, nuclear stainings in the polyploid nuclei of the peripodial membrane are easy to visualize and make appropriate positive controls for EcR stainings. The framing and the resolution of the images presented (which includes a full field of view taken with a 40X objective lens across all samples) was necessary for the proper confocal sectioning of just the ventral surface of the wing, the subject of the current investigation. Lower resolution would make it more difficult to isolate just the ventral signal from any signal present on the dorsal surface of the wing, which was not under investigation. Note that to provide this type of surface-specific data we performed left and right wing dissections separately to always be able to distinguish dorsal and ventral wing surfaces for each specimen examined.

Single-stage sampling for hormone titer comparisons doesn't make sense because critical periods are known to be different for different species. 20E titers only provided for a single timepoint in this study (wandering larvae), because this is the important stage for B. anynana. There are several important problems with this lack of temporal sampling. First of all, across insects the timing of hormone pulses and sensitive periods can be very specific and short-lived, and small changes in these pulses and periods are almost certainly significant for trait evolution. Therefore a single time point, generally, cannot serve as proxy for an entire endocrine process in a cross-species comparative study, at least without some sort of reference time series for each species. Second, this is almost certainly a problem in this study since it is known that the 20E pulse and sensitive period for color pattern polyphenism in J. coenia is at a later time point that is not even sampled in this paper (Rountree and Nijhout, 1995). I am concerned that by sampling only a single timepoint in all the species in this study, there may be important false negatives. Third, I think by not doing time series that there is simply a lot of interesting biology that is being missed. To me it is very interesting that both B. anynana and J. coenia express EcR in their eyespots (the latter observation is mostly ignored in this paper), yet have different sensitive periods and responses. Clearly there is more complex evolution going on here. I would like for this paper to talk more about this. Most importantly, though, they should have titer time series to have a complete, honest, and interesting story.

Yes, we agree with the reviewer that evolution of the critical time period for temperature sensitivity across species is a likely explanation for why other species do not exhibit the particular form of plasticity encountered in B. anynana. However, our goal was to try and examine how the specific pattern of plasticity in B. anynana evolved. We did not set out to explain how all patterns of plasticity that might be present across all these species evolved. This would be an unwieldy goal for a single study. Moreover, it is not possible for us to do additional time sampling at this stage due to experimental and funding limitations. We have modified our manuscript in specific sections to make our goals clearer and also mention that the critical time period for temperature sensitivity is likely evolving across species.

Staging all injection experiments at a single stage is not sufficient for comparative analysis because it is known that hormone-sensitive periods vary between species. All 20E and CucB injections were at a single stage (the wandering larva). This is problematic because many of the key conclusions of the paper are based on negative results from these injections. For many of the reasons outlined above, including previous experimental data from J. coenia, it is critical to do multiple timepoints. From these single time point injections we cannot rule out the possibility that the non-responsive species may simply be responsive at different time points. I am especially skeptical about V. dejone, which shows EcR expression in the eyespot and has a very strong eyespot temperature response. As I describe elsewhere, I am also skeptical that the black spots in I. leucone are homologous with B. anyana eyespots at all, so I am not sure how meaningful the results from this species are at all.

Yes, we agree with the reviewer that we cannot rule out the possibility that other species regulate their patterns of plasticity at different stages of development, but this is not what we set out to investigate. Again, we focused our investigation on the wandering stage, where there are definite temperature mediated differences in hormone titers and EcR signaling in Bicyclus anynana, the species around which we centered this investigation.

Odd inconsistency in injection controls. Shouldn't the "V" negative controls be the same between the 20E and CucB experiments? Yet they are extremely different, in some cases requiring completely different axis scales between experiments. In a few cases the differences between the different negative controls is much greater than the differences between the treatment and the negative control. The B. anyana results looks especially strange. Either I am missing something that needs to be explained better, or there was some problem with the experiments.

While “Vehicle” negative controls are indeed injected with the same ethanol-saline solution, we performed two batches of injections for the vehicle injected animals. One performed alongside the 20E injections, and another performed alongside the CucB injections. These two vehicle groups were kept separate for consistency. The small differences in “V” are due to the different batches of larvae used for the experiments.

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    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.

    elife-49544-fig1-data1.docx (16.6KB, docx)
    Figure 2—source data 1. 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.
    elife-49544-fig2-data1.docx (13.7KB, docx)
    Figure 3—source data 1. Mean body weight of wandering larvae, hemolymph volume and natural 20E titers at two different rearing temperatures; 20E and CucB injection volume.
    elife-49544-fig3-data1.docx (13.4KB, docx)
    Figure 4—source data 1. Results of likelihood ratio tests and AIC comparisons.

    See Figure 1—figure supplement 1 for node identities.

    elife-49544-fig4-data1.docx (13.2KB, docx)
    Transparent reporting form
    elife-49544-transrepform.docx (245.9KB, docx)

    Data Availability Statement

    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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