Single master regulatory gene coordinates the evolution and development of butterfly color and iridescence Linlin Zhang, Anyi Mazo-Vargas, and Robert D. Reed Edited by Sean B. Carroll, Howard Hughes Medical Institute and University of Wisconsin-Madison, Madison, WI, and approved August 1, 2017 (received for review May 31, 2017) Abstract The optix gene has been implicated in butterfly wing pattern adaptation by genetic association, mapping, and expression studies. The actual developmental function of this gene has remained unclear, however. Here we used CRISPR/Cas9 genome editing to show that optix plays a fundamental role in nymphalid butterfly wing pattern development, where it is required for determination of all chromatic coloration optix knockouts in four species show complete replacement of color pigments with melanins, with corresponding changes in pigment-related gene expression, resulting in black and gray butterflies. We also show that optix simultaneously acts as a switch gene for blue structural iridescence in some butterflies, demonstrating simple regulatory coordination of structural and pigmentary coloration. Remarkably, these optix knockouts phenocopy the recurring "black and blue" wing pattern archetype that has arisen on many independent occasions in butterflies. Here we demonstrate a simple genetic basis for structural coloration, and show that optix plays a deeply conserved role in butterfly wing pattern development. Sign up for PNAS alerts. Get alerts for new articles, or get an alert when an article is cited. Butterfly wing pattems provide an important model system for studying the interplay among ecological, developmental and genetic factors in the evolution of complex morphological traits. Dozens of genes have been implicated in wing pattem development thanks to a combination of comparative expression and, more recently, knockout studies (1-4) Interestingly, however, mapping and association work has highlighted only a small subset of these genes that seem to play a causative role in wing pattern adaptation in nature: optix, WA, cortex, and doublesex (5-10). These genes are particularly compelling for two reasons. First, they have all been genetically associated with local adaptation in multiple populations and/or species, and are thus characterized as "adaptive hotspot" genes that repeatedly drive morphological evolution across different lineages (11, 12). Second, based on detailed crossing and expression studies, we infer that these genes behave as complex trait regulators, with different alleles associated with different spatial expression domains that determine highly varied and complex color patterns, not simply the presence or absence of individual features. Although there is strong interest in these genes for these reasons, their specific developmental roles and the depth of conservation of their color patteming functions remain unclear. Here we present a comparative functional analysis of the optix gene in butterflies. This gene is linked to adaptive geographic variation of red ommochrome color patterns in the genus Heliconius, although its actual function remained unconfirmed before the present study (5, 13), opdx is also interesting because it is expressed in association with norpigmentation wing traits in various species, including morphologically derived wing conjugation scales, suggesting that it may have multiple regulatory roles in both wing scale coloration and structure (5, 14). In the present work, we used Cas9-mediated targeted deletion of optix to test its color patterning function in four species of nymphalid butterflies. Not only did we confirm deeply conserved roles for optix in coordinating pigmentation and scale morphology in all species surveyed, but we were surprised to find that this gene simultaneously regulates blue structural indescence in some butterflies. Importantly, this coordinated regulation of pigmentation and iridescence strongly phenocopies wing patterns seen in other distantly related species, leading us to hypothesize that optic may have played a role in wing pattern evolution in many different butterfly lineages. Results optix Simultaneously Represses Melanins and Promotes Ommochromes. optix was first identified as a wing potem gene candidate in Heliconius butterflies, in which mapping, association, and in situ expression data suggest a role in the determination of red color patterns (5, 14, 15). Subsequent mRNA-seq work also showed up-regulation of optix in red color patterns of the painted lady butterfly Vanessa cardul, raising the possibility of a more widespread role for this gene in red color pattem specification (16). To functionally confirm the role of optic in color patterning, we used a Cas9-mediated long-deletion mosaic knockout approach (4, 16, 17) in four nymphalid species: Heliconius erato, Agraulis vanillee, V. cardul, and Junonia coenia (Dataset S1, Tables S1 and S2). optix knockout in Herato produced results predicted by previous genetic and in situ hybridization studies. Mosaics revealed loss of the red color patterns previously shown to be presaged by pupal optix expression, including the color field at the base of the forewing (the so-called 'dennis" element) and the hindwing rays (Fig. 1A and B and Dataset S1, Tables S1 and S2). Not only was red pigmentation lost in knockout clones, but red pigments were replaced by black pigments. These results show that optix is required for red color pattem specification in Herato, and acts as a coordinating "or" switch between ormochrome (orange and red) and melanin (black and gray) pigment types. Fig. 1. optix determines wing scale color identity and morphology in H. erato and A. vanillae. (A) optix mosaic knockouts in H erato result in conversion of red ommochrome color pattems to black melanin. The comparisons shown are left-right asymmetrical knockout effects from single individual injected butterflies (0) Detail of mutant clone highlighted in the mutant in A showing red replaced by black in a proximal red "dennis" pattern of the dorsal forewing. (C-C) optix knockout mosaics showing transformation of pointed wing conjugation scales to normal wing scales. Each panel in the series shows successive detail. (D) optix replaces orange and brown ommochromes in A vanillee with melanins, resulting in a black and silver butterfly. Amows highlight presumptive clone boundaries discussed in the text. (E) Detail of a knockout clone boundary highlighting the switch between red and black pigmentation in the ventral forewing from D. (F) Ventral view of black spots in optix knockout mutant showing a phenotype similar to WT. (G and G) Wing conjugation scales in WT (G) and optik knockout mutant (G) demonstrating a role for optic in determining A. vanilla scale morphology. To test whether optix has a role in color patterning in a more basal heliconine butterfly, we generated knockouts in the gulf fritillary A. vanillae (Fig. 1D-F, Fig. S1, and Dataset S1, Tables S1 and S2). Previous in situ hybridization work in A producing a very unusual and dramatic phenotype of a completely black and silver butterfly (Fig. 10). We also observed a handful of orange or brown scales that changed to silver patches (Fig. 1D, ventral forewing, green arrows), although we cannot confidently conclude that these are cell-autonomous knockout effects since it has been shown that silver scales can be induced through long-range signaling (18, 19), in this case potentially from neighboring knockout clones. The wild- type (WT) black spots and marginal bands in the ventral forewing were unaffected in knockouts and remained a darker color relative to the neighboring mutant melanic scales (Fig. 1F), optic knockout also resulted in melanic hyperpigmentation in adult bodies (Fig. S14). Thus, our results in A. vanillae are consistent with those in H. erato in supporting a role for optix as a switch-like regulator that toggles between ommochrome and melanin patterns. Fig. 51. A. vanila WT and optic knockout phenotypes in developing wings and body. Ilustrative optic mosaic knockout phenotypes are shown at different stages of development (A) aduk. (B) ommochrome stage (when ommochrome pigments first appear). (C) melanin stage (when black melanin pigments first began to appear), and (D) late melanin stage (when black melanin pigments are prevalent across the wing). Arrowheads and dashed lines highlight illustrative mutant done boundaries. We next aimed to test whether optix regulates wing patterning in more distantly related lineages by performing knockouts in the nymphalines V. cardul (Fig. 2, Fig. S2, and Dataset S1, Tables S1 and S2) and J. coenia (Fig. 3, Fig. S3, and Dataset S1, Tables S1 and S2), which diverged from heliconines by 75-80 mya (20, 21). Our results were consistent with those from H. erato and A. vanillae, where optic knockouts in both species showed mutant clones with complete loss of presumptive ommochrome pigments and replacement by melanins (Figs. 2A-E and 3A-C). One interesting exception to this finding was in V. cardul, where the complete ormochrome-to-melanin switch consistently occurred in dorsal wings Fig. 2A and ), but much of the ventral wing area showed only a loss of omnochrome and little obvious hypermelanization (Fig. 2A, C, and D). Importantly, however, we recovered late-stage pupal wings from V. cardul that had phenotypes representing biallelic optix deletion clones. We have no direct evidence for this, however, given the challenges in rigorously characterizing specific alleles from individual mutant dones (6). We also recovered hypermarcopt scales, where it operates as an 'or function between ommochrome and melanin identities, but also may be modulated to serve as an "and" function in some contents, as demonstrated by phenotypes seen in the ventral wings of V. card Fig. 2 optix determines wing scale color identity and morphology in V. cardul (A) optix knockout mutant showing loss of cmmochrome pigments (0-0) Left-right asymmetrical comparisons from individual optix mutant butterflies, showing melanization of red pattems (B), loss of color pigmentation without widespread hypermelanization in the ventral forewing (C) and hindwing (D). (E) Severe defects in late-stage pupal wings displaying hypermetanization in red regions of dorsal and ventral wing surfaces (green and purple amowheads) compared with mosaic adult mutares in A. (F) optx knockout showing conversion of pointed wing conjugation scales to normal scales. Fig. 3. optix coordinates pigment color and structural descence in J. coenia. (A) optic knockout results in loss of red eyespot ring (6), replacement of orange ommochrome with presumptive melanin in ventral discal spot (DI) red patterns (C), and detail of knockout-induced iridescence on the ventral hindwing (D). (E) Mosaic knockout dones showing asymmetrical variation in promoted melanin and iridescence induction in dorsal forewings. (F) Wing conjugation scales in WT and optix knockout mutant Fig. 52. V. cardul WT and optix knockout phenotypes in developing wings and pupae. Mustrative optimes knockout phenotypes are shown at different stages of development (A) adult and pupa, (B) ommochrome stage (when omochrome pigments first appear). (C) melanin stage (when black melanin pigments first began to appear) and (D) late melanin stage (when black melanin pigments are prevalent across the wing). Arrowheads and dashed lines highlight ustrative mutant clone boundaries. The actual wings used for the RNA-seq experiments shown in Fig. 5 are labeled Fig. 53 J. conia WT and optik knockout phenotypes in developing wings and pupae. Ilustrative optix mosaic knockout phenotypes are shown at different stages of development (A) whole adults and pupa, (B) details of clone boundaries on adult wings showing indescent and nonridescent color shifts, and (C) late melanin stage wings (when black melanin pigments are prevalent across the wing). Arrowheads and dashed lines highlight illustrative mutant clones and boundaries. The actual wings used for RNA-seq experiments in Fig. 5 are labeled "RNA-seq optix Function Is Required for Determination of Derived Scale Structures. Along with its expression in color patterns, in situ optx expression also precisely predicts the location of patches of derived, pointed scales thought to play a role in conjugating forewings and hindwings during fight (51). To determine whether optix plays a role in determining the unusual morphology of these scales, we examined optic knockouts for changes in wing scale structure. Indeed, we found that in all four species, optix knockout resulted in transformation of wing conjugation scales to normal wing scales (Figs. 1 C and G, 2, and 3). Furthermore, in H. erato, A. vanilla and V card, where wing conjugation scales display color pigmentation, we observed both structural and pigmentation changes in the same scales, suggesting that optix can coregulate both scale morphology and pigmentation simultaneously. One final observation of note relates to the opti-expressing pheroscales that occur along the veins of male A. van (14) These scales did not show any grossly apparent transformation in optix knockouts (data not shown), even though the scales occured within obvious knockout clones. Therefore, whether optix plays a functional role in the development of pheroscales, as was predicted previously (14), remains an open question. In sum, our observations that optix knockout results in transformation of wing conjugation scales to normal wing scales indicate that optik plays a deeply conserved role in switching between discrete, complex scale morphologies in butterfly wings. optix Regulates Iridescence in J. coenia. The most surprising results from the present study came from our work in J. coenix, where knockout of optix induced strong blue iridescence in wing scales (Fig. 3A-E and Fig. S3). This induction of structural color occurred in addition to the loss of ormochrome pigmentation described above. Broad, strong indescence occurred in knockouts across dorsal wing surfaces, including in scales that are normally buff or orange in WT butterflies, such as the bright-orange discals spots and eyespot rings (Fig. 3A and B) and the marginal bands of the dorsal hindwing (Fig. 3A). Iridescence induction was less pronounced on v Iridescence was least a st apparent in was of the wing they clearly occured (Fig. 3A and D). It distal tip of the f in WT individuals, such as the black b and b k rings around the eyespots i strong ommock ceps (Ag 34 and e transition, we also recovered some c a partial v transformation in v which ormochromes were replaced by p d by presumptive melanins, but lacked iridescence (Fig. 3). These individuals often also showed additional mutant clones with iridescence, thus ruling out a background transregulatory effect. We speculate that these clones may represent k lower dosage effects due to clones being monoallelic for deletions; however, we have not confirmed this hypothesis. In sum, our knockout experiments in J. conia show that optix is a repressor of structural iridescence in this species, and that this regulatory function of optix occurs in addition to its other functions in pigment regulation. J. coenia optix Mutants Phenocopy Distantly Related Species. One striking aspect of the J. coenia optix knockout phenotype is the degree to which it phenocopies the archetypal "black and bluewing pattems that seem to have continually recured in many distantly related species across all butterfly families. Even simply focusing on the nymphalid tribe Junonin, which includes J. coenia, phylogenetic analysis suggests multiple origins of predominantly black and blue wing patterns as both fload phenotypes and plastic seasonal variants (Fig. 4). Notable examples of fixed black and blue phenotypes are se sare seen in such species as Junonia ataxia, which are almost indistinguishable from J. c coenia optix knockout phenotypes on casual observation (Fig. 4). Along w with closely phenocopying other species, optix knockouts are also: o strikingly reminiscent of seasonal phenotypes in such butterfies as Precis octavia (Fig. 40), in which the wet season form is predominantly red-orange and the dry season form is an archetypal black and blue phenotype with highly reduced red pattems. These seasonal color pattern differences might be explained by local changes in optix expression, although further work is needed to test this hypothesis. In sum, optix knockout phenotypes show many striking perallels with natural interspecific variation, leading us to speculate that differences in optix expression may be responsible for much of the wing pattern diversity seen in nymphalid butterflies Fig. 4. History of indescence in Junonia and related butterfly genera. (A) J. coenia optix knockouts phenocopy other junenne species of the black and blue pattern archetype. (B) The iridescent dry season form of P. octavia is largely consistent with the optix knockout effects seen in J. coenia. (C) Parsimony reconstruction of indescence in Junonin suggests multiple origins as a fixed phenotype. Species highlighted in red are shown in A. Prevalence of iridescence across dorsal wing surfaces is color coded where the light green dark blue continuum represents the approximate proportion of the wing surface that is indescent (Materials and Methods) Global Expression Profiling of Butterfly Wings in Response to optix Knockout. To investigate how wing pattems are controlled by optix, we used RNA-seq to compare transcript abundance in WT and optix knockout wings of V cardul and J. coenia. We sampled forewings and hindwings separately at a late stage of pupal development when ommochrome and melanin pigments are visible, in two biological replicates of both WT and strong knockout phenotypes (Figs. 52 and S3 and Dataset S1, Table S3). We first examined the expression of optix itself and confirmed a significant depletion of optix transcripts in all knockout wings (Dataset S1, Table S4). A closer analysis of opti transcript reads failed to reveal any partial transcripts showing lesions at the Cas9 cut site, suggesting that mutant transcripts resulting from edited alleles do not persist in wing tissue. We then aimed to identity all highly differentially expressed genes (DEG) in comparisons between WT and optix knockout wings using cutoff values of a fold change of >4 and a false discovery rate (FOR) of <0.001 in V. cardul, 97 unigenes were up-regulated and 243 were down-regulated in optix knockout wings compared with WT wings. We noted that Gene Ontology (GO) terms related to "structural constituent of cuticle were significantly enriched in optix knockouts, while organ morphogenesis" and "transport were down-regulated (Dataset S1, Table S5). In J. coena, only 31 unigenes were significantly up-regulated and 37 were down- regulated in optix knockouts. As in V. cardul, opdix knockout down-regulated transcripts related primarily to cellular transport Meanwhile, transcripts related to "muscle thin filament assembly were enriched in optix knockouts (Dataset 51, Table S5). To identify pigmentation genes potentially regulated by the optic network, we sorted for transcripts that show differential expression in optix knockout vs. WT wings and are orthologs or paralogs of putative pigmentation genes expressed during pigment maturation and/or spatially associated with red and black color regions in V. cardw (1). Using these criteria, we identified 12 genes associated with onmochrome pigmentation and 3 genes associated with melanin pigmentation in V card (Fig. 5 and Dataset S1, Tables S4 and 5). We found that Drosophila ommochrome pathway genes cinnabar and kynurenine formamidase (k) showed significant down-regulation in optix mutants. F Four unigenes coding for ommochrome-associated transporters were also down-regulated, including three major facilitator superfamily (ms) s and one A ATP-binding cassette transporter C family member. Another strongly by down-regulated transcript was juvenile hormone binding protein (HBP), a gene of unknown function that showed one of the strongest signals of red color association in a previous study (16). Knockouts also showed strong down-regulation of several melanin pathway genes, including tan, ebony, and yellow-d. Of note, all three of these genes are involved in the synthesis of N-B-alan dopamine sclerodin, which produces yellowish-tan hues. Fig. 5. RNA-seq analysis reveals differential gene expression in response to optix knockout. (A) Volcano plots of individual gene expression levels with log-twofold change (x-axis) against P value (FDR, y-axis, exact test) in cardul and J. conia DEGS (log-wofold 22, P<0.01) are in red. (8) Expression levels of candidate pigmentation and scale structure genes across replicate WT and optic knockout V. cardul and J. ocenia pupal wings. Our DEG results in J. coenia overlap with those of V. cardul in many areas, but also include several different transcripts (Fig. 5 and Dataset S1, Tables S4 and S7). In J. coenia, we identified nine ommochrome-associated genes, including cinnabar and if and five mis transporter transcripts that showed down-regulation in optix knockouts importantly, the 2 transcript appears to be orthologous between V. cardu and J. conia, suggesting that this may be a conserved the two ommochrome synthesis gene in butterflies. The melanin-associated genes were somewhat different between the b instead of yellow-d, two other yellow gene family members, yellow-x and yellow- x knockout wings. Surprisingly, two transcripts belonging to the JBP superior difrontally expressed superfamly showed dramatic up-regulation in optix knockout mutants. Of note, these two JHP transcripts are not orthologs of the ormochrome-associated genes in V carow and s d showed relatively low expression levels fragments per kilobase of transcript per million mapped fragments (FPKM) during wing development. In sum, these transcriptomic comparisons show that optix directly or indirectly 대 regulates a sizable suite of downstream genes during butterfly wing development, and both positively and negatively regulates distinct batteries of pigmentation genes, consistent with its role in switching between ormochrome and melanin pigment pattems. Furthermore, our data suggest that there may be differences in the downstream targets of the opti network between species, suggesting that downstream interactions in the pathway have diverged. While a fair amount is known about the genetic basis of insect pigmentation, virtually nothing is known about the types of genes that may control structural coloration. Thus we wanted to use our experimental system to identify potential candidate genes that may underle butterfly indescence. This task is more challenging than identifying pigmentation genes, because there are few precedents for how to informatically highlight DEGs that may have a role in determining scale ture. Recent work identified actin filaments as being indescence-producing scale generation and regulation Dataset S1, 1857 In indescent opknockoutsed in mentor outs (22) dus le s we scanned our DEG chain a we found significant down-regulation of two F-actin filament organization-related genes, myosin n2 and thioredoxin, and a cuticle-related gene, larval cuticle protein A28. We We also noted strong up-regulation in k in knockouts of 2-4-3-3 epsion, which has been shown to be involved in Ras/MAP kinase pathway and Drosophila eye development (23). Dataset S1, Table S7 highlights other candidates as well. A number of these genes are interesting candidates for effectors of iridescence; however, they should be considered preliminary candidates until further functional work confirms their roles. Nonetheless, we now have an experimental system in which we can modulate iridescence by knocking out a single gene, making uncovering the gene regulatory networks underlying structural coloration a more tractable problem. the comparative transcription work were forged de nove in the wings or were carried with optix from some ancestral role elsewhere in the insect, perhaps the omochrome-bearing eyes (). Whatever the case, with optix we now have a case study of a switch-like regulator gene that can be deployed anywhere in an organism to toggle between multiple discrete color states, and that has also played a role in color pater evolution in multiple species (24, 25). The stage is now set for asking a deeper set of questions about how an adaptive hotspot gene can gain novel functions over time, and what kind historical and mechanistic phenomena might drive it to play a repeated rolle in morphological evolution Materials and Methods CRISPR/Cas9 Genome Editing. We opted to generate long deletions using dual sRNAs following the protocol of Zhang and Reed (4, 16, 17, 33, SURNA target sequences were identified by searching for GGNING or N2ONGG patems targeting the optix exon and then tested for uniqueness by BLAST against the genome or transcriptome reference (Dataset S1, Table S1). Target regions were amplified by genotyping primers flanking the target regions, gel-purified, subcloned into a TOPO TA vector (Invitrogen), and sequenced (Fig. S4 and Dataset S1, Table S1). Fig. S4 sRNA design and sequence genotyping of optik knockout mutants in H. erato (4). A. vanillae (B), V. card (C), and J. conia (D) Locations of sRNAs are shown relative to the predicted functional domain single-exon optix coding region. Sequences of optix alleles from the knockout mutants shown in the main figures confirm lesions at target sites. Purple indicates sRNA targets; red, PAM sequences; green, novel sequences not observed in WT alleles. Phylogenetic Analysis. The latest available phylogeny of Junonin (34) was used to estimate the pairs and losses in butterfly wing iridescence. Ancestral states were mapped using maximum parsimony in Mesquite (35). Specimens from the Comell University Insect Collection and specimen photos from Encyclopedia of Life were used to score iridescence levels in Junonini butterflies as a character. We first divided butterfly wings into four regions based on a nymphalid grand plan model basal, central border symmetry system, and discal spot (also called DI and Dil patterns). We further classified the indescence trait into five distinct levels-0, 1/4, 1/2, 34, and 1-depending on the extent of iridescence occurrence in those four regions (3) For example, J. orithya was counted as 1/2 because iridescence is well represented in two of the four defined domains (Le, central and border symmetry regions). Pupal Wing Isolation and mRNA Extraction. V. cardul and J. conia forewings and hindwings were rapidly dissected and stored in later (Lile Technologies) at -80 "C. Wings from melanin stages were then selected for RNA sequencing RNA isolation was performed using the Ambion Purelink RNA Mini Kit (Life Technology). Two biological replicates were sampled from both forewing and hindwing in both WT and optiknockout mutants, resulting in eight samples for each species. Asymmetrical major and minor mosaic effect forewings from a V. cardul optix knockout individual were further collected as two samples to take advantage of asymmetrical mosaic information. In summary, 10 RNA-seq samples were collected in V. cardul, and a RNA-seq samples were collected in J. conia Library construction and sequencing were conducted as described previously (15) Analysis of Transcript Expression Data. The V. card transcriptome assembly (1) was downloaded from www.butertygenome.org and served as a reference. To build a reference for conia RNA-seq analysis, sequencing reads from this study and National Center for Ditechnology Information's Gene Expression Omnibus (GEO) database (accession no. GSE401) were merged. Assembly was built using Trinity (7) after in silico normalization. The TransDecoder predicted geneset was then searched against spect Plan, and GO databases for gene, domain, and GO annousion, respectively. Sequencing data were subjected to quality control by removing PCR primers, adapters, and low-quality reads. Clean reads were further aligned with reference genes with Bowle2 (38) Gene expression levels were calculated using FPKM. Differential gene expression was calculated for comparisons between WT and optix knockout mutants based on edge R using cutoffs of a fold change of 22 and an FDR of 0.001 Only DEGs shared between forewing and hindwing in WT vs. mutant comparisons were kept. In V. cardul DEGs were further filtered with their expression levels of fold change 21.2 between large and small asymmetrical mosaicism in the same individual. V. card and coenia wing RNA-seq raw sequencing data, full transcriptome assembly, and expression profiles are available in the GEO database (accession no. GSE98678). Single master regulatory gene coordinates the evolution and development of butterfly color and iridescence Linlin Zhang, Anyi Mazo-Vargas, and Robert D. Reed Edited by Sean B. Carroll, Howard Hughes Medical Institute and University of Wisconsin-Madison, Madison, WI, and approved August 1, 2017 (received for review May 31, 2017) Abstract The optix gene has been implicated in butterfly wing pattern adaptation by genetic association, mapping, and expression studies. The actual developmental function of this gene has remained unclear, however. Here we used CRISPR/Cas9 genome editing to show that optix plays a fundamental role in nymphalid butterfly wing pattern development, where it is required for determination of all chromatic coloration optix knockouts in four species show complete replacement of color pigments with melanins, with corresponding changes in pigment-related gene expression, resulting in black and gray butterflies. We also show that optix simultaneously acts as a switch gene for blue structural iridescence in some butterflies, demonstrating simple regulatory coordination of structural and pigmentary coloration. Remarkably, these optix knockouts phenocopy the recurring "black and blue" wing pattern archetype that has arisen on many independent occasions in butterflies. Here we demonstrate a simple genetic basis for structural coloration, and show that optix plays a deeply conserved role in butterfly wing pattern development. Sign up for PNAS alerts. Get alerts for new articles, or get an alert when an article is cited. Butterfly wing pattems provide an important model system for studying the interplay among ecological, developmental and genetic factors in the evolution of complex morphological traits. Dozens of genes have been implicated in wing pattem development thanks to a combination of comparative expression and, more recently, knockout studies (1-4) Interestingly, however, mapping and association work has highlighted only a small subset of these genes that seem to play a causative role in wing pattern adaptation in nature: optix, WA, cortex, and doublesex (5-10). These genes are particularly compelling for two reasons. First, they have all been genetically associated with local adaptation in multiple populations and/or species, and are thus characterized as "adaptive hotspot" genes that repeatedly drive morphological evolution across different lineages (11, 12). Second, based on detailed crossing and expression studies, we infer that these genes behave as complex trait regulators, with different alleles associated with different spatial expression domains that determine highly varied and complex color patterns, not simply the presence or absence of individual features. Although there is strong interest in these genes for these reasons, their specific developmental roles and the depth of conservation of their color patteming functions remain unclear. Here we present a comparative functional analysis of the optix gene in butterflies. This gene is linked to adaptive geographic variation of red ommochrome color patterns in the genus Heliconius, although its actual function remained unconfirmed before the present study (5, 13), opdx is also interesting because it is expressed in association with norpigmentation wing traits in various species, including morphologically derived wing conjugation scales, suggesting that it may have multiple regulatory roles in both wing scale coloration and structure (5, 14). In the present work, we used Cas9-mediated targeted deletion of optix to test its color patterning function in four species of nymphalid butterflies. Not only did we confirm deeply conserved roles for optix in coordinating pigmentation and scale morphology in all species surveyed, but we were surprised to find that this gene simultaneously regulates blue structural indescence in some butterflies. Importantly, this coordinated regulation of pigmentation and iridescence strongly phenocopies wing patterns seen in other distantly related species, leading us to hypothesize that optic may have played a role in wing pattern evolution in many different butterfly lineages. Results optix Simultaneously Represses Melanins and Promotes Ommochromes. optix was first identified as a wing potem gene candidate in Heliconius butterflies, in which mapping, association, and in situ expression data suggest a role in the determination of red color patterns (5, 14, 15). Subsequent mRNA-seq work also showed up-regulation of optix in red color patterns of the painted lady butterfly Vanessa cardul, raising the possibility of a more widespread role for this gene in red color pattem specification (16). To functionally confirm the role of optic in color patterning, we used a Cas9-mediated long-deletion mosaic knockout approach (4, 16, 17) in four nymphalid species: Heliconius erato, Agraulis vanillee, V. cardul, and Junonia coenia (Dataset S1, Tables S1 and S2). optix knockout in Herato produced results predicted by previous genetic and in situ hybridization studies. Mosaics revealed loss of the red color patterns previously shown to be presaged by pupal optix expression, including the color field at the base of the forewing (the so-called 'dennis" element) and the hindwing rays (Fig. 1A and B and Dataset S1, Tables S1 and S2). Not only was red pigmentation lost in knockout clones, but red pigments were replaced by black pigments. These results show that optix is required for red color pattem specification in Herato, and acts as a coordinating "or" switch between ormochrome (orange and red) and melanin (black and gray) pigment types. Fig. 1. optix determines wing scale color identity and morphology in H. erato and A. vanillae. (A) optix mosaic knockouts in H erato result in conversion of red ommochrome color pattems to black melanin. The comparisons shown are left-right asymmetrical knockout effects from single individual injected butterflies (0) Detail of mutant clone highlighted in the mutant in A showing red replaced by black in a proximal red "dennis" pattern of the dorsal forewing. (C-C) optix knockout mosaics showing transformation of pointed wing conjugation scales to normal wing scales. Each panel in the series shows successive detail. (D) optix replaces orange and brown ommochromes in A vanillee with melanins, resulting in a black and silver butterfly. Amows highlight presumptive clone boundaries discussed in the text. (E) Detail of a knockout clone boundary highlighting the switch between red and black pigmentation in the ventral forewing from D. (F) Ventral view of black spots in optix knockout mutant showing a phenotype similar to WT. (G and G) Wing conjugation scales in WT (G) and optik knockout mutant (G) demonstrating a role for optic in determining A. vanilla scale morphology. To test whether optix has a role in color patterning in a more basal heliconine butterfly, we generated knockouts in the gulf fritillary A. vanillae (Fig. 1D-F, Fig. S1, and Dataset S1, Tables S1 and S2). Previous in situ hybridization work in A producing a very unusual and dramatic phenotype of a completely black and silver butterfly (Fig. 10). We also observed a handful of orange or brown scales that changed to silver patches (Fig. 1D, ventral forewing, green arrows), although we cannot confidently conclude that these are cell-autonomous knockout effects since it has been shown that silver scales can be induced through long-range signaling (18, 19), in this case potentially from neighboring knockout clones. The wild- type (WT) black spots and marginal bands in the ventral forewing were unaffected in knockouts and remained a darker color relative to the neighboring mutant melanic scales (Fig. 1F), optic knockout also resulted in melanic hyperpigmentation in adult bodies (Fig. S14). Thus, our results in A. vanillae are consistent with those in H. erato in supporting a role for optix as a switch-like regulator that toggles between ommochrome and melanin patterns. Fig. 51. A. vanila WT and optic knockout phenotypes in developing wings and body. Ilustrative optic mosaic knockout phenotypes are shown at different stages of development (A) aduk. (B) ommochrome stage (when ommochrome pigments first appear). (C) melanin stage (when black melanin pigments first began to appear), and (D) late melanin stage (when black melanin pigments are prevalent across the wing). Arrowheads and dashed lines highlight illustrative mutant done boundaries. We next aimed to test whether optix regulates wing patterning in more distantly related lineages by performing knockouts in the nymphalines V. cardul (Fig. 2, Fig. S2, and Dataset S1, Tables S1 and S2) and J. coenia (Fig. 3, Fig. S3, and Dataset S1, Tables S1 and S2), which diverged from heliconines by 75-80 mya (20, 21). Our results were consistent with those from H. erato and A. vanillae, where optic knockouts in both species showed mutant clones with complete loss of presumptive ommochrome pigments and replacement by melanins (Figs. 2A-E and 3A-C). One interesting exception to this finding was in V. cardul, where the complete ormochrome-to-melanin switch consistently occurred in dorsal wings Fig. 2A and ), but much of the ventral wing area showed only a loss of omnochrome and little obvious hypermelanization (Fig. 2A, C, and D). Importantly, however, we recovered late-stage pupal wings from V. cardul that had phenotypes representing biallelic optix deletion clones. We have no direct evidence for this, however, given the challenges in rigorously characterizing specific alleles from individual mutant dones (6). We also recovered hypermarcopt scales, where it operates as an 'or function between ommochrome and melanin identities, but also may be modulated to serve as an "and" function in some contents, as demonstrated by phenotypes seen in the ventral wings of V. card Fig. 2 optix determines wing scale color identity and morphology in V. cardul (A) optix knockout mutant showing loss of cmmochrome pigments (0-0) Left-right asymmetrical comparisons from individual optix mutant butterflies, showing melanization of red pattems (B), loss of color pigmentation without widespread hypermelanization in the ventral forewing (C) and hindwing (D). (E) Severe defects in late-stage pupal wings displaying hypermetanization in red regions of dorsal and ventral wing surfaces (green and purple amowheads) compared with mosaic adult mutares in A. (F) optx knockout showing conversion of pointed wing conjugation scales to normal scales. Fig. 3. optix coordinates pigment color and structural descence in J. coenia. (A) optic knockout results in loss of red eyespot ring (6), replacement of orange ommochrome with presumptive melanin in ventral discal spot (DI) red patterns (C), and detail of knockout-induced iridescence on the ventral hindwing (D). (E) Mosaic knockout dones showing asymmetrical variation in promoted melanin and iridescence induction in dorsal forewings. (F) Wing conjugation scales in WT and optix knockout mutant Fig. 52. V. cardul WT and optix knockout phenotypes in developing wings and pupae. Mustrative optimes knockout phenotypes are shown at different stages of development (A) adult and pupa, (B) ommochrome stage (when omochrome pigments first appear). (C) melanin stage (when black melanin pigments first began to appear) and (D) late melanin stage (when black melanin pigments are prevalent across the wing). Arrowheads and dashed lines highlight ustrative mutant clone boundaries. The actual wings used for the RNA-seq experiments shown in Fig. 5 are labeled Fig. 53 J. conia WT and optik knockout phenotypes in developing wings and pupae. Ilustrative optix mosaic knockout phenotypes are shown at different stages of development (A) whole adults and pupa, (B) details of clone boundaries on adult wings showing indescent and nonridescent color shifts, and (C) late melanin stage wings (when black melanin pigments are prevalent across the wing). Arrowheads and dashed lines highlight illustrative mutant clones and boundaries. The actual wings used for RNA-seq experiments in Fig. 5 are labeled "RNA-seq optix Function Is Required for Determination of Derived Scale Structures. Along with its expression in color patterns, in situ optx expression also precisely predicts the location of patches of derived, pointed scales thought to play a role in conjugating forewings and hindwings during fight (51). To determine whether optix plays a role in determining the unusual morphology of these scales, we examined optic knockouts for changes in wing scale structure. Indeed, we found that in all four species, optix knockout resulted in transformation of wing conjugation scales to normal wing scales (Figs. 1 C and G, 2, and 3). Furthermore, in H. erato, A. vanilla and V card, where wing conjugation scales display color pigmentation, we observed both structural and pigmentation changes in the same scales, suggesting that optix can coregulate both scale morphology and pigmentation simultaneously. One final observation of note relates to the opti-expressing pheroscales that occur along the veins of male A. van (14) These scales did not show any grossly apparent transformation in optix knockouts (data not shown), even though the scales occured within obvious knockout clones. Therefore, whether optix plays a functional role in the development of pheroscales, as was predicted previously (14), remains an open question. In sum, our observations that optix knockout results in transformation of wing conjugation scales to normal wing scales indicate that optik plays a deeply conserved role in switching between discrete, complex scale morphologies in butterfly wings. optix Regulates Iridescence in J. coenia. The most surprising results from the present study came from our work in J. coenix, where knockout of optix induced strong blue iridescence in wing scales (Fig. 3A-E and Fig. S3). This induction of structural color occurred in addition to the loss of ormochrome pigmentation described above. Broad, strong indescence occurred in knockouts across dorsal wing surfaces, including in scales that are normally buff or orange in WT butterflies, such as the bright-orange discals spots and eyespot rings (Fig. 3A and B) and the marginal bands of the dorsal hindwing (Fig. 3A). Iridescence induction was less pronounced on v Iridescence was least a st apparent in was of the wing they clearly occured (Fig. 3A and D). It distal tip of the f in WT individuals, such as the black b and b k rings around the eyespots i strong ommock ceps (Ag 34 and e transition, we also recovered some c a partial v transformation in v which ormochromes were replaced by p d by presumptive melanins, but lacked iridescence (Fig. 3). These individuals often also showed additional mutant clones with iridescence, thus ruling out a background transregulatory effect. We speculate that these clones may represent k lower dosage effects due to clones being monoallelic for deletions; however, we have not confirmed this hypothesis. In sum, our knockout experiments in J. conia show that optix is a repressor of structural iridescence in this species, and that this regulatory function of optix occurs in addition to its other functions in pigment regulation. J. coenia optix Mutants Phenocopy Distantly Related Species. One striking aspect of the J. coenia optix knockout phenotype is the degree to which it phenocopies the archetypal "black and bluewing pattems that seem to have continually recured in many distantly related species across all butterfly families. Even simply focusing on the nymphalid tribe Junonin, which includes J. coenia, phylogenetic analysis suggests multiple origins of predominantly black and blue wing patterns as both fload phenotypes and plastic seasonal variants (Fig. 4). Notable examples of fixed black and blue phenotypes are se sare seen in such species as Junonia ataxia, which are almost indistinguishable from J. c coenia optix knockout phenotypes on casual observation (Fig. 4). Along w with closely phenocopying other species, optix knockouts are also: o strikingly reminiscent of seasonal phenotypes in such butterfies as Precis octavia (Fig. 40), in which the wet season form is predominantly red-orange and the dry season form is an archetypal black and blue phenotype with highly reduced red pattems. These seasonal color pattern differences might be explained by local changes in optix expression, although further work is needed to test this hypothesis. In sum, optix knockout phenotypes show many striking perallels with natural interspecific variation, leading us to speculate that differences in optix expression may be responsible for much of the wing pattern diversity seen in nymphalid butterflies Fig. 4. History of indescence in Junonia and related butterfly genera. (A) J. coenia optix knockouts phenocopy other junenne species of the black and blue pattern archetype. (B) The iridescent dry season form of P. octavia is largely consistent with the optix knockout effects seen in J. coenia. (C) Parsimony reconstruction of indescence in Junonin suggests multiple origins as a fixed phenotype. Species highlighted in red are shown in A. Prevalence of iridescence across dorsal wing surfaces is color coded where the light green dark blue continuum represents the approximate proportion of the wing surface that is indescent (Materials and Methods) Global Expression Profiling of Butterfly Wings in Response to optix Knockout. To investigate how wing pattems are controlled by optix, we used RNA-seq to compare transcript abundance in WT and optix knockout wings of V cardul and J. coenia. We sampled forewings and hindwings separately at a late stage of pupal development when ommochrome and melanin pigments are visible, in two biological replicates of both WT and strong knockout phenotypes (Figs. 52 and S3 and Dataset S1, Table S3). We first examined the expression of optix itself and confirmed a significant depletion of optix transcripts in all knockout wings (Dataset S1, Table S4). A closer analysis of opti transcript reads failed to reveal any partial transcripts showing lesions at the Cas9 cut site, suggesting that mutant transcripts resulting from edited alleles do not persist in wing tissue. We then aimed to identity all highly differentially expressed genes (DEG) in comparisons between WT and optix knockout wings using cutoff values of a fold change of >4 and a false discovery rate (FOR) of <0.001 in V. cardul, 97 unigenes were up-regulated and 243 were down-regulated in optix knockout wings compared with WT wings. We noted that Gene Ontology (GO) terms related to "structural constituent of cuticle were significantly enriched in optix knockouts, while organ morphogenesis" and "transport were down-regulated (Dataset S1, Table S5). In J. coena, only 31 unigenes were significantly up-regulated and 37 were down- regulated in optix knockouts. As in V. cardul, opdix knockout down-regulated transcripts related primarily to cellular transport Meanwhile, transcripts related to "muscle thin filament assembly were enriched in optix knockouts (Dataset 51, Table S5). To identify pigmentation genes potentially regulated by the optic network, we sorted for transcripts that show differential expression in optix knockout vs. WT wings and are orthologs or paralogs of putative pigmentation genes expressed during pigment maturation and/or spatially associated with red and black color regions in V. cardw (1). Using these criteria, we identified 12 genes associated with onmochrome pigmentation and 3 genes associated with melanin pigmentation in V card (Fig. 5 and Dataset S1, Tables S4 and 5). We found that Drosophila ommochrome pathway genes cinnabar and kynurenine formamidase (k) showed significant down-regulation in optix mutants. F Four unigenes coding for ommochrome-associated transporters were also down-regulated, including three major facilitator superfamily (ms) s and one A ATP-binding cassette transporter C family member. Another strongly by down-regulated transcript was juvenile hormone binding protein (HBP), a gene of unknown function that showed one of the strongest signals of red color association in a previous study (16). Knockouts also showed strong down-regulation of several melanin pathway genes, including tan, ebony, and yellow-d. Of note, all three of these genes are involved in the synthesis of N-B-alan dopamine sclerodin, which produces yellowish-tan hues. Fig. 5. RNA-seq analysis reveals differential gene expression in response to optix knockout. (A) Volcano plots of individual gene expression levels with log-twofold change (x-axis) against P value (FDR, y-axis, exact test) in cardul and J. conia DEGS (log-wofold 22, P<0.01) are in red. (8) Expression levels of candidate pigmentation and scale structure genes across replicate WT and optic knockout V. cardul and J. ocenia pupal wings. Our DEG results in J. coenia overlap with those of V. cardul in many areas, but also include several different transcripts (Fig. 5 and Dataset S1, Tables S4 and S7). In J. coenia, we identified nine ommochrome-associated genes, including cinnabar and if and five mis transporter transcripts that showed down-regulation in optix knockouts importantly, the 2 transcript appears to be orthologous between V. cardu and J. conia, suggesting that this may be a conserved the two ommochrome synthesis gene in butterflies. The melanin-associated genes were somewhat different between the b instead of yellow-d, two other yellow gene family members, yellow-x and yellow- x knockout wings. Surprisingly, two transcripts belonging to the JBP superior difrontally expressed superfamly showed dramatic up-regulation in optix knockout mutants. Of note, these two JHP transcripts are not orthologs of the ormochrome-associated genes in V carow and s d showed relatively low expression levels fragments per kilobase of transcript per million mapped fragments (FPKM) during wing development. In sum, these transcriptomic comparisons show that optix directly or indirectly 대 regulates a sizable suite of downstream genes during butterfly wing development, and both positively and negatively regulates distinct batteries of pigmentation genes, consistent with its role in switching between ormochrome and melanin pigment pattems. Furthermore, our data suggest that there may be differences in the downstream targets of the opti network between species, suggesting that downstream interactions in the pathway have diverged. While a fair amount is known about the genetic basis of insect pigmentation, virtually nothing is known about the types of genes that may control structural coloration. Thus we wanted to use our experimental system to identify potential candidate genes that may underle butterfly indescence. This task is more challenging than identifying pigmentation genes, because there are few precedents for how to informatically highlight DEGs that may have a role in determining scale ture. Recent work identified actin filaments as being indescence-producing scale generation and regulation Dataset S1, 1857 In indescent opknockoutsed in mentor outs (22) dus le s we scanned our DEG chain a we found significant down-regulation of two F-actin filament organization-related genes, myosin n2 and thioredoxin, and a cuticle-related gene, larval cuticle protein A28. We We also noted strong up-regulation in k in knockouts of 2-4-3-3 epsion, which has been shown to be involved in Ras/MAP kinase pathway and Drosophila eye development (23). Dataset S1, Table S7 highlights other candidates as well. A number of these genes are interesting candidates for effectors of iridescence; however, they should be considered preliminary candidates until further functional work confirms their roles. Nonetheless, we now have an experimental system in which we can modulate iridescence by knocking out a single gene, making uncovering the gene regulatory networks underlying structural coloration a more tractable problem. the comparative transcription work were forged de nove in the wings or were carried with optix from some ancestral role elsewhere in the insect, perhaps the omochrome-bearing eyes (). Whatever the case, with optix we now have a case study of a switch-like regulator gene that can be deployed anywhere in an organism to toggle between multiple discrete color states, and that has also played a role in color pater evolution in multiple species (24, 25). The stage is now set for asking a deeper set of questions about how an adaptive hotspot gene can gain novel functions over time, and what kind historical and mechanistic phenomena might drive it to play a repeated rolle in morphological evolution Materials and Methods CRISPR/Cas9 Genome Editing. We opted to generate long deletions using dual sRNAs following the protocol of Zhang and Reed (4, 16, 17, 33, SURNA target sequences were identified by searching for GGNING or N2ONGG patems targeting the optix exon and then tested for uniqueness by BLAST against the genome or transcriptome reference (Dataset S1, Table S1). Target regions were amplified by genotyping primers flanking the target regions, gel-purified, subcloned into a TOPO TA vector (Invitrogen), and sequenced (Fig. S4 and Dataset S1, Table S1). Fig. S4 sRNA design and sequence genotyping of optik knockout mutants in H. erato (4). A. vanillae (B), V. card (C), and J. conia (D) Locations of sRNAs are shown relative to the predicted functional domain single-exon optix coding region. Sequences of optix alleles from the knockout mutants shown in the main figures confirm lesions at target sites. Purple indicates sRNA targets; red, PAM sequences; green, novel sequences not observed in WT alleles. Phylogenetic Analysis. The latest available phylogeny of Junonin (34) was used to estimate the pairs and losses in butterfly wing iridescence. Ancestral states were mapped using maximum parsimony in Mesquite (35). Specimens from the Comell University Insect Collection and specimen photos from Encyclopedia of Life were used to score iridescence levels in Junonini butterflies as a character. We first divided butterfly wings into four regions based on a nymphalid grand plan model basal, central border symmetry system, and discal spot (also called DI and Dil patterns). We further classified the indescence trait into five distinct levels-0, 1/4, 1/2, 34, and 1-depending on the extent of iridescence occurrence in those four regions (3) For example, J. orithya was counted as 1/2 because iridescence is well represented in two of the four defined domains (Le, central and border symmetry regions). Pupal Wing Isolation and mRNA Extraction. V. cardul and J. conia forewings and hindwings were rapidly dissected and stored in later (Lile Technologies) at -80 "C. Wings from melanin stages were then selected for RNA sequencing RNA isolation was performed using the Ambion Purelink RNA Mini Kit (Life Technology). Two biological replicates were sampled from both forewing and hindwing in both WT and optiknockout mutants, resulting in eight samples for each species. Asymmetrical major and minor mosaic effect forewings from a V. cardul optix knockout individual were further collected as two samples to take advantage of asymmetrical mosaic information. In summary, 10 RNA-seq samples were collected in V. cardul, and a RNA-seq samples were collected in J. conia Library construction and sequencing were conducted as described previously (15) Analysis of Transcript Expression Data. The V. card transcriptome assembly (1) was downloaded from www.butertygenome.org and served as a reference. To build a reference for conia RNA-seq analysis, sequencing reads from this study and National Center for Ditechnology Information's Gene Expression Omnibus (GEO) database (accession no. GSE401) were merged. Assembly was built using Trinity (7) after in silico normalization. The TransDecoder predicted geneset was then searched against spect Plan, and GO databases for gene, domain, and GO annousion, respectively. Sequencing data were subjected to quality control by removing PCR primers, adapters, and low-quality reads. Clean reads were further aligned with reference genes with Bowle2 (38) Gene expression levels were calculated using FPKM. Differential gene expression was calculated for comparisons between WT and optix knockout mutants based on edge R using cutoffs of a fold change of 22 and an FDR of 0.001 Only DEGs shared between forewing and hindwing in WT vs. mutant comparisons were kept. In V. cardul DEGs were further filtered with their expression levels of fold change 21.2 between large and small asymmetrical mosaicism in the same individual. V. card and coenia wing RNA-seq raw sequencing data, full transcriptome assembly, and expression profiles are available in the GEO database (accession no. GSE98678).