Hypothetical Illustration of Gene Network Co-Option and De Novo Network Evolution

<p>(A) Modular gene network where gene <i>X</i>, at the top of a regulatory network, directs expression of gene <i>Y</i>, which in turn directs expression of gene <i>Z</i>. All these genes are expressed in the tip of an appendage (e.g., leg) depicted on the right. (B) The modular gene network is co-opted into a new tissue by the evolution of a novel CRE in gene <i>X</i>. The <i>Y</i> and <i>Z</i> genes, which only receive inputs from <i>X</i> and <i>Y</i> genes, respectively, are also turned on in the new tissue (e.g., eyespot centers in a butterfly larval wing). The CREs of the <i>Y</i> and <i>Z</i> genes now have a dual function in directing gene expression in two separate developmental contexts, e.g., they are pleiotropic. (C) De novo network evolution where elements of the same non-modular gene network, <i>X</i>, <i>Y</i>, and <i>Z</i>, each evolve a separate CRE that drives gene expression in the novel developmental context.</p

Seasonal form and mating status influence frequency and latency to copulation.

<p>Effect of seasonal form and male and female mating status on <b>a)</b> frequency of copulation, and <b>b)</b> latency to copulation for pairs of WS and DS butterflies. Panels on the left illustrate the effect of female mating status on frequency of and latency to copulation in the mixed sex pairs. Panels on the right illustrate the effect of male mating status on frequency of and latency to copulation. Seasonal form and female mating status were the two significant parameters in GLMs for frequency of copulation and latency to copulation. Non-virgin females have lower copulation frequency and higher latency to copulation than virgin females, but this effect is not present in males. DS forms of each sex display higher copulation frequency and lower latency to copulation than WS forms. Asterisk (*) indicates p<0.01.</p

Examples of Pleiotropic CREs

<div><p>A schematic representation of putatively pleiotropic CREs is shown for: (A) The <i>spalt</i> (<i>sal</i> and <i>salr</i>) gene complex; (B) <i>spineless</i> (<i>ss</i>); (C) <i>yellow</i> (<i>y</i>); (D) <i>odd-skipped</i> (<i>odd</i>). Gene orientation is marked by arrows. Ovals show approximate position of CREs surrounding the protein-coding genes. Checkmarks of tissue/organs above CREs represent the multiple domains of gene expression driven by the same CRE. Modified from [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1000037#pbio-1000037-b024" target="_blank">24</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1000037#pbio-1000037-b026" target="_blank">26–28</a>]. The multiple CREs that drive gene expression in the same tissue or organ mostly drive gene expression in distinct cell populations.</p> <p>Abbreviations: CNS, central nervous system; PNS, peripheral nervous system.</p></div

Schematic of the different temperature shift experiments performed in this study.

<p>Each temperature shift treatment has been assigned a different letter, and experiments are grouped by specific question and analysis (Exp 1–3). L, P, and A stand for larval, pupal, and adult development, respectively. Brown (the color of dead leaf litter) indicates the DS temperature of 17°C and green (the color of lush vegetation) represents the WS temperature of 27°C.</p

Variation in wing patterning of WS and DS <i>B</i>. <i>anynana</i> butterflies.

<p>Here we show a DS female and WS male mating in the laboratory. The DS female is the upper left of the two mating butterflies, while the WS male is the lower right butterfly. DS butterflies have smaller ventral eyespots than WS butterflies, and a more mottled brown patterning on the ventral surfaces of their wings. While wing size is sexually dimorphic in this species, the seasonal variation in eyespot size and general ventral wing surface coloration is sexually monomorphic.</p

Additional file 7: Figure S2. of Wound healing, calcium signaling, and other novel pathways are associated with the formation of butterfly eyespots

Crosses between wild-type and Spotty butterflies. Female Wt individuals were crossed with Spotty male individuals. All F1 (first generation) offspring had phenotypes associated with wt/Spotty heterozygous and displayed small intermediate eyespots in sectors M2 and M3. These F1 individuals were mated with each other to produce a F2 generation. Only wild-type and Spotty homozygous individuals were selected from the F2 generation and these were mated to individuals of the same phenotype to produce two pure-breeding F3 generation cohorts. Female and male wild-type and Spotty pupae from this generation were used for RNA extractions. (TIFF 860 kb

Similar Gene Expression in Various Non-Homologous Structures

<p>The transcription factor Distal-less (Dll) is expressed (A) in the horn primordium of the African dung beetle, Onthophagus nigriventris(modified from [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1000037#pbio-1000037-b009" target="_blank">9</a>]); (B) in the eyespot focus of the squinting bush brown butterfly, Bicyclus anynana (modified from [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1000037#pbio-1000037-b040" target="_blank">40</a>]); and (C) in the leg imaginal disc of the fruit fly, D. melanogaster (modified from [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1000037#pbio-1000037-b041" target="_blank">41</a>]). The transcription factor Spalt (Sal) is expressed (D) in the antenna imaginal disc of D. melanogaster (modified from [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1000037#pbio-1000037-b042" target="_blank">42</a>]); and (E) in the eyespot field of B. anynana pupal wings (modified from [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1000037#pbio-1000037-b012" target="_blank">12</a>]).</p

WS, but not DS individuals incur a longevity cost when mating with non-virgins instead of virgins.

<p>Visualization of the interactive effect of seasonal form and partner’s mating status on life span (data for males and females combined). Star signifies p = 0.021 for interaction between seasonal form and partner’s mating status. Asterisk (*) indicates p<0.01 for mating status of mate.</p

Non-virgin WS males, but not DS males, increase their time in copula relative to that of virgin males.

<p>Visual representation of the main factors in the GLM of time in copula. <b>a)</b> Effect of seasonal form and female mating status on copulation duration (female mating status does not alter time in copula for either seasonal form). <b>b)</b> Effect of seasonal form and male mating status on copulation duration. Star signifies p = 0.025 for interaction between mating status and rearing environment. Asterisk (*) indicates p<0.01.</p

Supplementary Materials from <i>apterous A</i> specifies dorsal wing patterns and sexual traits in butterflies

Butterflies have evolved different colour patterns on their dorsal and ventral wing surfaces to serve different signalling functions, yet the developmental mechanisms controlling surface-specific patterning are still unknown. Here, we mutate both copies of the transcription factor <i>apterous</i> in <i>Bicyclus anynana</i> butterflies using CRISPR/Cas9 and show that <i>apterous A,</i> expressed dorsally, functions both as a repressor and modifier of ventral wing colour patterns, as well as a promoter of dorsal sexual ornaments in males. We propose that the surface-specific diversification of wing patterns in butterflies proceeded via the co-option of <i>apterous A</i> or its downstream effectors into various gene regulatory networks involved in the differentiation of discrete wing traits. Further, interactions between <i>apterous</i> and sex-specific factors such as <i>doublesex</i> may have contributed to the origin of sexually dimorphic surface-specific patterns. Finally, we discuss the evolution of eyespot number diversity in the family Nymphalidae within the context of developmental constraints due to <i>apterous</i> regulation