Alternative mating tactics, mutual mate choice, and multiple mating cues can contribute to reproductive isolation but are rarely all addressed in case studies of speciation. Here, we dissected the behavioral components of assortative mating using the closely related parapatric butterflies Heliconius erato cyrbia and Heliconius himera. We tested male and female preference, the role of geography, the use of color or pheromones in female choice, and male choice in pupal mating, and an alternative mating tactic. We found evidence of female preference for conspecific males in both species, while only H. erato males showed evidence of courtship preference. Female H. himera rejected males with blocked androconial pheromones over controls but did not show bias against males with altered coloration. In the context of pupal mating, an alternative mating strategy, we observed only H. erato males were less likely to make interspecies mating mistakes. This work underscores the importance of collecting behavioral data to the study of speciation and highlights how multiple traits can contribute to reproductive isolation even between closely related species....

Transcription factors (TF) are critical for development and cellular processes and are found in all organisms. How their DNA-binding specificity changes through time has yet to be fully understood. TF DNA-binding specificity is determined by how their DNA-binding domain (DBD) interacts with DNA. TFs are identified by the sequence homology shared with described DBDs, which allows them to be classified into families. It is accepted that similar DBDs have the same DNA-binding specificity and bind to the same sequences. However, changes in a TF can lead to changes in its DNA recognition. TFs members of the sine oculis homeobox (SIX) homeodomain family are found from sponges to humans and are considered atypical members of the homeodomain (HD) family. They regulate numerous processes and phenotypic features, from eye development in flies and humans to red color patterning in Heliconius butterflies wings to human brain development. How evolutionary related TFs diversify has yet to be fully understood, especially diversification of their DNA-binding specificity. To understand the evolutionary history of this family, we performed phylogenetic inference that placed the first SIX within Porifera and the presence of the three canonical SIX (sine oculis, optix, and six4) in Cnidaria. In addition, we observe the presence of two major groups that show that optix and six4 are more evolutionary related. To determine changes in DNA-binding specificity, we performed in vitro Systematic Evolution of Ligands by Exponential Enrichment (SELEX-seq) using full length SIX TF proteins from Drosophila melanogaster, Heliconius erato, and Homo sapiens. Our data shows the majority of SIX TFs bind to the canonical binding motif (5' -TGATAC-3' ), except for six4 members, which seem to prefer (5' -TGACAC-3' ).<br /> <br /> Interestingly, the way they bind to these motifs differs. Both sine oculis and six4 homologs require a 5' -GA dinucleotide flanking the core motif on the 5' -end. In comparison, optix related members prefer a shorter flaking region and less dependence on 5' -GA. This is interesting since optix is more evolutionarily related to six4 than to sine oculis. We also found that Heliconius erato optix can bind DNA both as a monomer and as a homodimer with a preferred spacing of 2-bp between binding sites. Using the determined DNA-binding specificity of optix, we were able to predict optix binding to cis-regulatory elements (CRE) active during wing development. optix was capable to bind to all the predicted sites, including to its own promoter. Validation of optix binding to these CREs allows to expand the search of optix gene targets and contribute to our understanding of the mechanism of wing development and red color patterns in Heliconius butterflies....

Butterfly wing patterns have captivated evolutionary and developmental biologists for decades because of their vast diversity. Studies have focused on the molecular basis of pattern variation in the Nymphalidae, with little effort put toward skippers, family Hesperiidae. This study had two aims: identify the presence of Wnt genes through ISH and pharmacological drug injections, and test differentially expressed genes in three developmental stages, between wing types, and between wing compartments of E. clarus using RNA-sequencing. I hypothesized, that a simple yet modular set of genes was involved in wing pattern development of the skipper, similar to that of nymphalid butterflies and that genes such as WntA and Ubx would be differentially expressed as in nymphalids. ISH confirmed the presence of two Wnt genes and RNA-seq provided evidence of differential expression, suggesting that the genes underlining wing pattern diversity are being shared between these two distantly related families....