Macroevolutionary shifts of WntA function potentiate butterfly wing-pattern diversity

Macroevolutionary shifts of WntA function potentiate

butterfly wing-pattern diversity

Anyi Mazo-Vargas

, Carolina Concha

b,1

, Luca Livraghi

c,1

, Darli Massardo

d,1

, Richard W. R. Wallbank

b,e,1

, Linlin Zhang

Joseph D. Papador

, Daniel Martinez-Najera

, Chris D. Jiggins

b,e

, Marcus R. Kronforst

, Casper J. Breuker

Robert D. Reed

, Nipam H. Patel

f,g

, W. Owen McMillan

, and Arnaud Martin

h,2

Department of Ecology and Evolutionary Biology, Cornell University, Ithaca, NY 14853;

Smithsonian Tropical Research Institute, Gamboa, Panama;

Department of Biological and Medical Sciences, Oxford Brookes University, Oxford OX3 0BP, United Kingdom;

Department of Ecology and Evolution,

University of Chicago, Chicago, IL 60637;

Department of Zoology, University of Cambridge, Cambridge CB2 3EJ, United Kingdom;

Department of

Integrative Biology, University of California, Berkeley, CA 94720;

Department of Molecular Cell Biology, University of California, Berkeley, CA 94720;

and

Department of Biological Sciences, The George Washington University, Washington, DC 20052

Edited by Sean B. Carroll, Howard Hughes Medical Institute and University of Wisconsin–Madison, Madison, WI, and approved August 7, 2017 (received for

review May 16, 2017)

Butterfly wing patterns provide a rich comparative framework to

study how morphological complexity develops and evolves. Here

we used CRISPR/Cas9 somatic mutagenesis to test a patterning

role for WntA, a signaling ligand gene previously identified as a

hotspot of shape-tuning alleles involved in wing mimicry. We

show that WntA loss-of-function causes multiple modifications

of pattern elements in seven nymphalid butterfly species. In three

butterflies with a conserved wing-pattern arrangement, WntA is

necessary for the induction of stripe-like patterns known as sym-

metry systems and acquired a novel eyespot activator role specific

to Vanessa forewings. In two Heliconius species, WntA specifies

the boundaries between melanic fields and the light-color patterns

that they contour. In the passionvine butterfly Agraulis, WntA re-

moval shows opposite effects on adjacent pattern elements, re-

vealing a dual role across the wing field. Finally, WntA acquired a

divergent role in the patterning of interveinous patterns in the

monarch, a basal nymphalid butterfly that lacks stripe-like symme-

try systems. These results identify WntA as an instructive signal for

the prepatterning of a biological system of exuberant diversity

and illustrate how shifts in the deployment and effects of a single

developmental gene underlie morphological change.

Wnt signaling

pattern formation

evolutionary tinkering

gene co-opt ion

CRISPR mutagenesis

he multitude of patterns found in developing organisms is

achieved by a small number of conserved signaling pathways,

which raises an important question. How does biodiversity arise

from the sharing of constituents across a single tree of life? One

explanation for this apparent paradox is that conserved regulatory

genes evolve new “tricks” or roles during development (1).

Assessing this phenomenon requires comparing the function of

candidate genes across a dense phylogenetic sampling of divergent

phenotypes. Here, the patterns on butterfly wings provide an ideal

test case. The development of scale-covered wings, their structural

and pigment comp lexity, and an elaborate patterning system are

key features of the Lepidoptera (moths and butterflies), which

form about 10% of all species known to humankind (2). Wing

patterns across the group are fantastically diverse and are often

shaped by natural and sexual selection (3). Studies in fruit flies,

butterflies, and moths have implicated secreted Wnt-signaling li-

gands as color pattern inducers (4–8). In butterfly wings, two lines

of evidence suggest a prominent patterning role for the Wnt ligand

gene WntA in particular. First, WntA was repeatedly mapped as a

locus driving pattern-shape adaptations involved in mimicry, and

a total of 18 WntA causative alleles have been identified across a

wide phylogenetic spectrum (9–13). Second, WntA expression

marks developing wing domains that prefigure the position and

shape of pattern elements of various color compositions (10, 14).

The nymphalid groundplan provides a conceptual framework

to understand pattern variation in butterflies (3). Under this

framework, patterns are organized into parallel subdivisions of

autonomous color pattern complexes known as “symmetr y sys-

tems,” which are arranged across the dorsal and ventral surfaces of

both the fore- and hindwing (14–19) (Fig. 1 A–C). This arrange-

ment is thought to represent a putative archetype of a butterfly

wing pattern, and diversity is created by modifying elements within

and among these symmetry systems (3). WntA is typically expressed

in three of the four symmetry systems (14): the small proximal

pattern called Basalis (B), the large median pattern called the

Central Symmetry System (CSS), and the Marginal Band System

(MBS), which features laminar stripes bordering the wing. Here we

used CRISPR/Cas9 mutagenesis to impair WntA function and as-

sess its patterning roles in Nymphalidae, the largest butterfly family

that radiated around 90 Mya (20). We characterize the de-

velopmental function of WntA in species representative of the

nymphalid groundplan and then show that WntA has acquired di-

vergent patterning roles in several lineages.

Results and Discussion

We injected Cas9/sgRNA duplexes into 1–6 h butterfly embryos

at a syncytial stage (n = 5,794 eggs). As only a fraction of the

dividing nuclei are edited, the resulting mosaicism can bypass the

deleterious effects of developmental mutations and yields G

escapers that survive until the adult stage for phenotypic analysis

(21–23). We performed CRISPR injections in seven nymphalid

Significance

Our study assesses the long-held hypothesis that evolution of

new gene functions underlies the diversification of animal forms.

To do this, we systematically compared the patterning roles of a

single gene across seven butterfly species. Under a null hypothesis

of gene stasis, each knockout experiment should yield directly

comparable phenotypes. We instead observed a varied repertoire

of lineage-specific effects in different wing regions, demonstrat-

ing that the repeated modification of a key instructive signal was

instrumental in the complex evolution of wing color patterns.

These comparative data confirm the heuristic potential of CRISPR

mutagenesis in nontraditional model organisms and illustrate the

principle that biodiversity can emerge from the tinkering of ho-

mologous genetic factors.

Author contributions: C.D.J. , M.R.K., C.J.B., R.D.R., N.H.P., W.O.M., and A.M. designed

research; A.M.-V., C.C., L.L., D.M., R.W.R.W., L.Z., J.D.P., D.M.-N., and A.M. performed

research; and A.M. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

C.C., L.L., D.M., and R.W.R.W. contributed equally to this work.

To whom correspondence should be addressed. Email: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.

1073/pnas.1708149114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1708149114 PNAS

October 3, 2017

vol. 114

no. 40

10701–10706

EVOLUTION

Fig. 1. WntA loss-of-function effects in groundplan-like nymphalids. (A–C) The nymphalid groundplan consists of consecutive symmetry systems organized

along the antero-posterior axis. Color code indicates groundplan elements in subsequent panels. Orange: Baso-Discal Complex (BDC) patterns; blue: CSS;

fuchsia: Bordel Ocelli Symmetry System (BoSS), including dPf; green: MBS, including Sub-Marginal Band (SMB). Dots show wing topological landmarks cor-

responding to vein crossings. (D–G) WntA mKO in J. coenia results in the loss of WntA

patterns. (D) Whole-wing phenotypes. (E) In situ hybridization of WntA

in WT fifth instar imaginal disks. (F) Blow-up of proximal forewing area showing the loss of B upon WntA mKO. (G) Blow-up of proximal forewing area

showing the distalization of dPf and SMB elements. (H–K) Replication of the J. coenia result s in P. aegeria.(H) Whole-wing phenotypes. (I) In situ hybridization

of WntA in WT fifth instar imaginal disks. (J) Loss of the forewing CSS. (K) Distalization of dark-brown dPF and SMB; arrowheads point at corresponding WntA

expression domains in I.

10702

www.pnas.org/cgi/doi/10.1073/pnas.1708149114 Mazo-Vargas et al.

species to induce frameshift mutations in WntA-coding exons.

About 10% of hatchlings (240 of 2,293 survivors) yielded adult

butterflies with mosaic knockout (mKO) pattern defects on their

wings (SI Appendix, Figs. S1–S9 and Tables S1 and S2).

WntA Induces Central Symmetry Systems. First we used CRISPR to

test the effects of WntA loss-of-function on the wing patterns of the

Common Buckeye Junonia coenia (tribe: Junoniini). WntA mKOs

resulted in a complete loss of the CSS, consistent with WntA ex-

pression that prefigures its shape and position in the wing imaginal

disks (Fig. 1 D and E and SI Appendix,Fig.S1). The WntA-positive

forewing B element was lost while the wg-positive D

-D

elements (8,

24) were unaffecte d (Fig. 1F). The B-D

-D

patterns have a similar

color composition, indicating that WntA and wg play interchangeable

roles in their induction. In contrast, the double loss of the dis-

tinct B and CSS patterns also illustrates the regional specificity of

WntA-signaling color outputs across the wing surface. In the

marginal section of the wing (Fig. 1G), WntA mKOs resulted in a

contraction of the MBS and in a shift of chevron patterns known

as the distal parafocal elements (dPF) (17, 19). WntA may impact

these distal elements by participating in complex patterning dy-

namics in the marginal section of the wing (25).

Variations on the WntA Groundplan Theme. Next we asked if the

instructive roles of WntA were phylogenetically conserved, using

two other nymphalid butterflies with a groundplan organization,

the Specked Wood Pararge aegeria (tribe: Satyrini) (15) and the

Painted Lady Vanessa cardui (tribe: Nymphalini) (18). WntA

Fig. 2. Conserved and novel aspects of WntA function in Painted Lady butterflies. (A and B) WntA mKOs in V. cardui result in defects or loss of the CSS,

highlighted in cyan in B.(C and D) Wnt A forewing expression is associated with the CSS (magnified in D) and the MBS in WT fifth instar wing disks. (E)Blow-

up of a CSS section showing pattern disruption upon WntA mKO. (F and G) WntA hindwing expression in the CSS and the MBS (magnified in G; pt, peripheral

tissue) in WT fifth instar wing disks. (H) WntA mKO results in distal shifts of dPf elements. (I) Blow-up of WntA expression in the hindwing CSS. (J) Magni-

fication of intermediate and severe levels of CSS reduction observed upon WntA mKO. ( K ) WntA expression as observed in the presumptive forewing

eyespots in late fifth instar win g disks of V. cardui.(L and M) Reduction of dorsal forewing eyespots following WntA mKO. (N) Color change in ventral mKO

forewing eyespots.

Mazo-Vargas et al. PNAS

October 3, 2017

vol. 114

no. 40

10703

EVOLUTION

mKOs yielded consistent effects by eliminating the CSS and

distalizing the parafocal elements in these two species (Figs. 1

H–K and 2 A–H and SI Appendix, Fig. S10). Of note, in the

V. cardui hindwing, the complex wave-like patterns of the CSS

were lost upon severe WntA mKO and reduced in more inter-

mediate forms (Fig. 2 I and J). These two species also highlighted

other aspects of WntA phenotypic effects. In P. aegeria hindwings,

the mKO-mediated disruption of the marginal system resulted

in an apparent expansion of the eyespot outer rings (SI Appendix,

Fig. S10D). V. cardui WntA mKOs resulted in the reduction of

each dorsal forewing eyespot (P values < 10

−4

;Fig.2K–M)and

generated color composition defects in the ventral forewing eye-

spots (Fig. 2N and SI Appendix,Fig.S3). Only V. cardui forewings

areknowntoexpressWntA in their eyespots (14). We thus infer

that WntA was co-opted in the eyespot gene regulatory network of

the V. cardui lineage to elaborate upon the patterning of this

complex feature (26). Overall, comparisons in three species show

that multifaceted modulations of WntA function have shaped var-

iations on the basic nymphalid groundplan theme.

WntA Induces Pattern Boundaries in Heliconius. We next focused

on species that departed more markedly from the nymphalid

Fig. 3. Variegated WntA loss-of-function phenotypes in passionvine butterflies. ( A– E) Effects of WntA mKO in H. e. demophoon.(A) Whole wings.

(B) Detection of WT proximal WntA expression by larval forewing in situ hybridization (zone 1). (C) Loss of proximal pattern boundary in WntA-positive zone

1. (D) Antero-proximal expression of WntA in WT late larval hindwings. (E) Loss of antero-proximal pattern boundary in mKO hindwings. (F–J) Effects of WntA

mKO in H. sara.(F) Whole wings. (G) Detection of proxi mal (zone 1) and median (zone 2) WntA in larval forewings. (H) Loss of proximal (green line) and

median (fuchsia line) pattern boundaries resulting in loss of melanic identity in zones 1 and 2. (I) Antero-proximal expression of WntA in larval hindwings.

(J) Widespread antero-proximal color identity shift in mKO hindwings. (K–P) Effects of WntA mKO in A. vanillae.(K) Whole wings. (L) Silver-spot–related

expression of WntA in larval forewings and loss in mKO forewings. (Bottom) M3-A1 spot triad. (M and N ) Silver spot-related expression of WntA in larval

hindwings (M) and loss/reduction in mKO hindwings (N). (Top) M3-A1 spot complex. (O) Silver-spot pattern expansion in proximal mKO hindwings.

(P) Secondary expression of WntA in the proximal region of late larval hindwings. Colored dots: wing topological landmarks (vein crossings).

10704

www.pnas.org/cgi/doi/10.1073/pnas.1708149114 Mazo-Vargas et al.

groundplan configuration, starting with the hyperdiverse Heliconius

clade (tribe: Heliconiini). We performed CRISPR mKOs in Cen-

tral American morphs of two species, Heliconius erato demophoon

and Heliconius sara sara. WntA removalresultedinanexpansionof

light-color patterns in both cases (Fig. 3 A and F). In H. erato

demophoon, WntA expression marked melanic patches that con-

tour forewing red and hindwing yellow stripes (Fig. 3 B and D).

Predictably, its loss-of-function resulted in the loss of the corre-

sponding boundaries with black contours being replaced by ex-

pansions of red or yellow (Figs. 3 C and E). H. sara forewing disks

showed a proximal and central WntA expression domains that each

correspond to melanic fields that frame the signature yellow stripes

of this butterfly (Fig. 3G). Both melanic intervals were lost fol-

lowing WntA mKOs (Fig. 3H), yielding an almost uniformly yellow

forewing surface. Hindwings showed a similar effect with WntA

deficiency resulting in melanic-to-yellow switches in the antero-

proximal half of the wing (Fig. 3J). Interestingly, this treatment

also revealed a cryptic stripe of red patches. A similar phenotype is

observed in subspecies of H. sara, as well as in its sister species

Heliconius leucadia (SI Appendix,Fig.S11), suggesting that mod-

ulations of Wnt signaling could underlie these cases of natural

variation. Overall, these data support previous predictions that

groundplan elements such as the CSS can be homologized to what

form the apparent contours of Heliconius patterns (27–29). WntA is

best thought as a prepatterning factor that determines boundaries

between color fields, a view that is compatible with the re-

placement effects of mKOs, where WntA-deficient cells acquire the

color fate of the adjacent territory. This property may explain why

cis-regulatory tinkering of WntA expression seems to underlie the

repeated modification of color pattern shapes across this explosive

radiation (9–12), as it allows the coordinated modulation of color

fate on either side of a moving boundary.

Antagonistic Roles of WntA in Adjacent Patterns. Compared with

Heliconius, the closely related Gulf Fritillary butterfly (Agraulis

vanillae) has modified the nymphalid groundplan differently to

produce its distinctive wing pattern (27). Rather than continuous

stripes, A. vanillae shows dispersed silver spots of identical color

composition, each consisting of a core of highly reflective “mirror”

scales (30) and an outline of black scales. A subset of silver spots

express WntA or wg (14), and accordingly, all of the WntA

patterns

contracted or disappeared in WntA mKOs (Fig. 3 K–N and SI Ap-

pendix,Fig.S6). Among the wg

elements (forewing D

and D

only D

coexpressed WntA and was specifically reduced in WntA

mKOs (SI Appendix,Fig.S12), suggesting that silver spots respond to

overall Wnt dosage. WntA mKOs also resulted in a drastic expansion

of WntA-free (WntA

−

)patterns(Fig.3O). Importantly, butterflies

treated with exogenous heparin, a ligand-binding molecule with Wnt

gain-of-function effects (9, 14, 31, 32), showed the opposite outcome:

expanded WntA

and reduced WntA

−

patterns (14). These reverse

effects of CRISPR loss-of-function vs. heparin gain-of-function

suggest that WntA activates and represses two distinct sets of pat-

terns, and the repressed domain in fact shows a secondary wave of

WntA expression in late larval instar wing disks (Fig. 3P). This ob-

servation leads us to propose that the dual effect of WntA may be

due to a biphasic deployment, with a first wave of WntA pattern-

activating expression followed by an inhibitory event in the Wnt-

repressed territory. Testing this working model will require the

identification and expression profiling of WntA-signaling targets in

A. vanillae.

Repurposing of WntA in a Reduced Groundplan. Finally, we used the

lack of visible CSS in monarchs (Danaus plexippus; tribe:

Danaini) as an example of extreme divergence from the nym-

phalid groundplan. WntA lacked a CSS median stripe expression

as expected and was instead detected around the presumptive

veins, indicative of a potential role in the induction of vein-

dependent patterns (33). WntA mKO adults showed drastic

expansions of the white interveinous patterns (Fig. 4), which are

usually visible as thin outlines of the veins in WT ventral wings.

In addition, white dot elements that ornate the marginal region

expanded and fused following WntA mKO. Other WntA mKO

monarchs showed a small dorsal patch of ectopic interveinous

scales in the crossvein region, demonstrating maximal WntA

expression in hindwings (SI Appendix, Fig. S13). Consistent with

a Wnt loss-of-function, this mild phenotype was reproduced by

injection of dextran sulfate, a drug treatment that emulates Wnt

signal inhibition in other butterflies (14, 32) (SI Appendix, Fig.

S14). Overall, expression and functional data suggest that WntA

was again repurposed, in this case as a repressor of interveinous

white scales in the monarch lineage.

Lessons from Somatic CRISPR Phenotypes. Somatic mutagenesis

yielded loss-of-function data in the G

adults of seven butterfly

species, an achievement that would have been unrealistic in the

pre-CRISPR era. Experimental replication using various single-

guide RNA (sgRNA) targets ruled out a contribution of off-

target lesions, and genotyping experiments revealed a pre-

dominance of frameshift, presumably null WntA alleles (SI

Appendix, Figs. S8 and S9). Variations in clone size, allelic dos-

age, and the possible occurrence of hypomorphic mutations

could underlie complex cases of mosaicism, explaining the range of

observed effects (Fig. 2J and SI Appendix, Figs. S1–S7). Inferring

the allelic composition of wing mutant clones from their geno-

typing is complicated by the movement of insect wing epithelial

cells following adult emergence (34), as well as by the presence of

cell contaminants that are unlikely to underlie the pattern phenotype

(e.g., tracheal cells, neurons, hemocytes). We attempted the gener-

ation of germline mutations in V. cardui to bypass the experimental

limitations of somatic heterogeneity. Following the injection of a

single sgRNA targeting the WntA stop codon, we obtained an adult

female bearing a modification of the forewing CSS (SI Appendix,

Fig. S15). Six G

offsprings displayed the same phenotype and

were all heterozygous for a 16-bp indel mutation, resulting in a

C-terminal Cys-Asn-Stop → Gly-Ser-Arg-Stop editing of the pre-

dicted WntA protein. This allele was passed to a second generation

Fig. 4. WntA loss-of-function in monarch butterflies induces interveinous white

scales. (A and B) In situ detection of WntA in Danaus plexippus last larval instar

forewing (A) and hindwing disks (B). (C) Whole-wing phenotypes following

WntA mKO. All effects consist of expansion of white scale patterns. (D)Close-up

view of larval hindwing WntA interveinous expression in the periphery of tra-

cheal vein precursors. (E and F) Expansion of interveinous white scale fate in the

hindwing region corresponding to D. Colored dots: wing topological landmarks

(vein crossings).

Mazo-Vargas et al. PNAS

October 3, 2017

vol. 114

no. 40

10705

EVOLUTION

but was subsequently lost due to an episode of high mortality in

our stock. Nonetheless, this preliminary result illustrates the po-

tential of CRISPR to induce a variety of loss-of-function alleles,

which could be propagated via the germline for tackling future

developmental questions where mosaicism is a concern.

Conclusions. The Nymphalidae family comprises about 6,000 but-

terfly species, most of which can be identified by their wing patterns.

We used this system as a proxy of morphological evolution and

found that a single signal articulates its underlying complexity, as

shown by the variety of WntA mKO phenotypes obtained across

different wing regions and species. Our data highlight three major

results. First, WntA is associated with multiple pattern elements

within the same individual, including within the same wing surface,

e.g., both the adjacent Basalis and CSS patterns require WntA in

J. coenia forewings, despite distinct color compositions, whereas CSS

stripes often differ between wing surfaces (dorsal vs. ventral, fore-

wing vs. hindwing). Wnt signaling may combine with selector genes

that mark distinct wing domains to mediate these regional-specific

outputs within a single individual (24, 35). Second, spatial shifts in

WntA expression cause pattern-shape evolution, exemplified by the

multitude of species-specific manifestations of the CSS. Cis-regula-

tory variants of WntA (9–12), or alternatively, modulations of the

trans-regulatory landscape that controls WntA expression, may have

fashioned these macroevolutionary shifts. Finally, WntA evolves new

patterning functions. It was co-opted into forewing eyespot forma-

tion in the V. cardui lineage, evolved a localized pattern-inhibiting

role in A. vanillae, and was repurposed for the patterning of vein-

contouring markings in monarchs. In summary, WntA instructs the

formation of multiple wing-pattern elements in the nymphalid ra-

diation, demonstrating the importance of prepatterning processes in

the unfolding of complex anatomy. The versatility of this signaling

factor illustrates how the repeated tinkering of a developmental

gene can foster boisterous evolutionary change.

Experimental Procedures

Butterflies. Insect stock origins, rearing conditions, and oviposition host plants

are described in SI Appendix, Table S3.

In Situ Hybridizations. WntA cDNA sequences, cloned or amplified with

T7 overhang primers, were used as a templa te to synthesize digoxigenin-

labeled RNA probe as described previously (14, 36). Primers for amplification

of template DNA are shown in SI Appendix, Table S4. In situ hybridization of

imaginal discs from fifth instar larvae were performed as described (14).

Egg Injections. Butterfly eggs laid on host plant leaves were collected after 1–6h

(SI Appendix,TablesS2andS3). J. coenia and V. cardui eggs were then washed for

20–100 s in 5% benzalkonium chloride (Sigma-Aldrich), rinsed in water, and dried

in a desiccation chamber or by air ventilation for softening the chorion. To soften

and separate egg mass in H. sara, clumps were treated with a 1:20 dilution of

Milton sterilizing fluid (Procter and Gamble) for 4 min, rinsed with water, and

dried. Eggs were arranged on a double-sided adhesive tape or glued to a glass

slide, usually with the micropyle facing up. CRISPR mixtures containing pre-

assembled sgRNAs and recombinant Cas9 protein (PNA Bio) were injected, using

pulled quartz or borosilicate needles. The concentration of sgRNAs and

Cas9 varied between butterfly species and experiments (SI Appendix, Table S2).

Genotyping. DNA was extracted from wing muscles or single legs using the

Phire animal tissue direct PCR kit (Thermo Fisher Scientific), and amplified

using oligonucleotides flanking the sgRNAs target region (SI Appendix, Table

S2). PCR amplicons were gel-purified, subcloned into the pGEM-T Easy Vec-

tor System (Promega), and sequenced on an ABI 3730 sequencer.

ACKNOWLEDGMENTS. We thank Saad Arif, Nora Braak, Chris Day, Melanie

Gibbs, Jonah Heller, José Hermina-Perez, Colin Morrison, Oscar Paneso, Manu

Sanjeev, David Tian, Camille Tulure, Matthew Verosloff, and Hans Van Dyck for

assistance with rearing, injecting, dissecting, and imaging butterflies; Lawrence

Gilbert for sharing photographs and insights on Heliconius wing patterning;

and Karin van der Burg, Bernardo Clavijo, David Jaffe, James Lewis, and James

Mallet for providing access to genomic data. This work was supported by grants

from the NSF (Grants DGE-1650441, IOS-1354318, IOS-1557443, and IOS-

1452648); the NIH (Grant GM108626); the Leverhulme Trust (Grant RPG-2014-

167); the Pew Charitable Trust; a Nigel Groome PhD studentship (Oxford

Brookes University); and the Smithsonian Institution.

1. Carroll SB (2008) Evo-devo and an expanding evolutionary synthesis: A genetic theory

of morphological evolution. Cell 134:25–36.

2. Kristensen NP, Scoble MJ, Karsholt O (2007) Lepidoptera phylogeny and systematics:

The state of inventorying moth and butterfly diversity. Zootaxa 1668:699–747.

3. Nijhout HF (1991) The Development and Evolution of Butterfly Wing Patterns

(Smithsonian Institution, Washington, DC).

4. Werner T, Koshikawa S, Williams TM, Carroll SB (2010) Generation of a novel wing

colour pattern by the Wingless morphogen. Nature 464:1143–1148.

5. Yamaguchi J, et al. (2013) Periodic Wnt1 expression in response to ecdysteroid gen-

erates twin-spot markings on caterpillars. Nat Commun 4:1857.

6. Koshikawa S, et al. (2015) Gain of cis-regulatory activities underlies novel domains of

wingless gene expression in Drosophila. Proc Natl Acad Sci USA 112:7524–7529.

7. Özsu N, Chan QY, Chen B, Gupta MD, Monteiro A (2017) Wingless is a positive reg-

ulator of eyespot color patterns in Bicyclus anynana butterflies. Dev Biol, 10.1016/j.

ydbio.2017.06.030.

8. Martin A, Reed RD (2010) Wingless and aristaless2 define a developmental ground

plan for moth and butterfly wing pattern evolution. Mol Biol Evol 27:2864–2878.

9. Martin A, et al. (2012) Diversification of complex butterfly wing patterns by repeated

regulatory evolution of a Wnt ligand. Proc Natl Acad Sci USA 109:12632–12637.

10. Gallant JR, et al. (2014) Ancient homology underlies adaptive mimetic diversity across

butterflies. Nat Commun 5:4817.

11. Huber B, et al. (2015) Conservatism and novelty in the genetic architecture of ad-

aptation in Heliconius butterflies. Heredity (Edinb) 114:515–524.

12. Van Belleghem SM, et al. (2017) Complex modular architecture around a simple

toolkit of wing pattern genes. Nat Ecol Evol 1:0052.

13. Martin A, Courtier-Orgogozo V (2017) Morphological evolution repeatedly caused by

mutations in signaling ligand genes. Diversity and Evolution of Butterfly Wing Patterns:

An Integrative Approach, eds Sekimura T, Nijhout F (Springer Singapore, Singapore).

14. Martin A, Reed RD (2014) Wnt signaling underlies evolution and development of the

butterfly wing pattern symmetry systems. Dev Biol 395:367–378.

15. Schwanwitsch B (1935) Evolution of the wing-pattern in Palaearctic Satyridae: Pararge

and five other genera. Acta Zool 16:143–281.

16. Schwanwitsch B (1956) Color-pattern in Lepidoptera. Entomol Obozr 35:530–546.

17. Otaki JM (2012) Color pattern analysis of nymphalid butterfly wings: Revision of the

nymphalid groundplan. Zoolog Sci 29:568–576.

18. Abbasi R, Marcus JM (2015) Color pattern evolution in Vanessa butterflies (Nym-

phalidae: Nymphalini): Non-eyespot characters. Evol Dev 17:63–81.

19. Taira W, Kinjo S, Otaki JM (2015) The marginal band system in nymphalid butterfly

wings. Zoolog Sci 32:38–

46.

20. Wahlberg N, et al. (2009) Nymphalid butterflies diversify following near demise at the

Cretaceous/Tertiary boundary. Proc Biol Sci 276:4295–4302.

21. Perry M, et al. (2016) Molecular logic behind the three-way stochastic choices that

expand butterfly colour vision. Nature 535:280–284.

22. Zhang L, Reed RD (2016) Genome editing in butterflies reveals that spalt promotes

and Distal-less represses eyespot colour patterns. Nat Commun 7:11769.

23. Zhang L, et al. (2017) Genetic basis of melanin pigmentation in butterfly wings.

Genetics 205:1537–1550.

24. Carroll SB, et al. (1994) Pattern formation and eyespot determination in butterfly

wings. Science 265:109–114.

25. Nijhout HF (1990) A comprehensive model for colour pattern formation in butterflies.

Proc R Soc Lond B Biol Sci 239:81–113.

26. Monteiro A, Gupta MD (2016) Identifying coopted networks and causative mutations

in the origin of novel complex traits. Curr Top Dev Biol 119:205–226.

27. Nijhout HF, Wray GA (1988) Homologies in the color patterns of the genus Heliconius

(Lepidoptera, Nymphalidae). Biol J Linn Soc Lond 33:345–365.

28. Nijhout HF, Wray GA, Gilbert LE (1990) An analysis of the phenotypic effects of certain color

pattern genes in Heliconius (Lepidoptera, Nymphalidae). Biol J Linn Soc Lond 40:357–37 2.

29. Gilbert LE (2003) Adaptive novelty through introgression in Heliconius wing patterns:

Evidence for shared genetic “tool box” from synthetic hybrid zones and a theory of

diversification. Ecology and Evolution Taking Flight: Butterflies as Model Systems, eds

Boggs CL, Watt WB, Ehrlich PR (Univ. of Chicago Press, Chicago).

30. Dinwiddie A, et al. (2014) Dynamics of F-actin prefigure the structure of butterfly

wing scales. Dev Biol 392:404–418.

31. Binari RC, et al. (1997) Genetic evidence that heparin-like glycosaminoglycans are

involved in wingless signaling. Development 124:2623–2632.

32. Serfas MS, Carroll SB (2005) Pharmacologic approaches to butterfly wing patterning:

Sulfated polysaccharides mimic or antagonize cold shock and alter the interpretation

of gradients of positional information. Dev Biol 287:416–424.

33. Nijhout HF (2010) Molecular and physiological basis of colour pattern formation. Adv

Insect Physiol 38:219–265.

34. Tögel M, Pass G, Paululat A (2008) The Drosophila wing hearts originate from peri-

cardial cells and are essential for wing maturation. Dev Biol 318:29–37.

35. Rebeiz M, Patel NH, Hinman VF (2015) Unraveling the tangled skein: The evolution of

transcriptional regulatory networks in development. Annu Rev Genomics Hum Genet

16:103–131.

36. Carter J-M, Gibbs M, Breuker CJ (2015) Divergent RNA localisation patterns of ma-

ternal genes regulating embryonic patterning in the butterfly Pararge aegeria.

PLoS One 10:e0144471.

10706

www.pnas.org/cgi/doi/10.1073/pnas.1708149114 Mazo-Vargas et al.