Understanding the evolutionary origins and factors maintaining alternative life history strategies (ALHS) within species is a major goal of evolutionary research. While alternative alleles causing discrete ALHS are expected to purge or fix over time, one-third of the ~90 species of Colias butterflies are polymorphic for a female-limited ALHS called Alba. Whether Alba arose once, evolved in parallel, or has been exchanged among taxa is currently unknown. Using comparative genome-wide association study (GWAS) and population genomic analyses, we placed the genetic basis of Alba in time-calibrated phylogenomic framework, revealing that Alba evolved once near the base of the genus and has been subsequently maintained via introgression and balancing selection. CRISPR-Cas9 mutagenesis was then used to verify a putative cis-regulatory region of Alba, which we identified using phylogenetic foot printing. We hypothesize that this cis-regulatory region acts as a modular enhancer for the induction of the Alba ALHS, which has likely facilitated its long evolutionary persistence. 

The intriguing coloration and patterning of butterfly wings have served as a cornerstone for understanding complex phenotypes and gene regulatory network (GRN) evolution. Here we investigate the pteridine pigment biosynthesis pathway in Pieridae butterflies, which produces their characteristic white, yellow, and orange hues. In light of the widespread evolution of complex traits via recruitment of existing GRNs, we investigate both the mechanisms and evolutionary history of the use of pteridines in Pieridae wings. Utilizing CRISPR/Cas9-induced gene knockouts (KOs), we probed two genes, SPR and Purple, critical to pteridine biosynthesis. The pleiotropic consequences of these KOs provide us with insights into extended phenotypic context and use of pteridine beyond wing coloration.

Our results confirmed that both SPR and Purple regulate pteridine formation but challenge existing models of pteridine biosynthesis. We additionally observed that pteridine is not only involved in wing color, but all body scales, enhancing our understanding of pteridine function across adult tissues. Lastly, we found that the granule structure that contains the pteridine, and is unique to pteridine pigmentation, appears to be ubiquitous to all scale types as well as all major clades of Pierid butterflies, potentially indicating representing an evolutionary novelty for Pieridae. 

Sexual dimorphisms represent a source of phenotypic variation and result from differences in how natural and sexual selection act on males and females within a species. Identifying the genetic basis of dimorphism can be challenging, especially once it is fixed within a species. However, studying polymorphisms, even when fixed within a population, can provide insights into the genetic basis of sexual dimorphisms. In this study, we investigate the genetic basis of a regionally isolated sexual dimorphism in the wings of Pieris napi adalvinda, a subspecies of P. napi found in northernmost Scandinavia, where females exhibit heavily melanized wings. By using a combination of male and female informative crosses, genomic sequencing of melanic outliers, and a population genomic analysis with a new reference genome for the melanic morph, we demonstrate that the female-limited morph adalvinda is caused by a single dominant allele at an autosomal locus upstream of the gene cortex. This novel finding adds to the growing body of literature that connects repeated mutations in and near the cortex gene to the regulation of butterfly wing melanization, providing insights into the evolution of sexual dimorphisms and the recruitment of genes into monomorphic or sex-limited forms. This study thus highlights the significance of cortex as a basis for a female-limited trait and lays the foundation for future comparative analyses of dimorphism genetic underpinnings.

Insects have been key players in the assessments of biodiversity impacts of anthropogenically driven environmental change, including the evolutionary and ecological impacts of climate change. Populations of Edith’s Checkerspot Butterfly (Euphydryas editha) adapt rapidly to diverse environmental conditions, with numerous high-impact studies documenting these dynamics over several decades. However, studies of the underlying genetic bases of these responses have been hampered by missing genomic resources, limiting the ability to connect genomic responses to environmental change. Using a combination of Oxford Nanopore long reads, haplotype merging, HiC scaffolding followed by Illumina polishing, we generated a highly contiguous and complete assembly (contigs n = 142, N50 = 21.2 Mb, total length = 607.8 Mb; BUSCOs n = 5,286, single copy complete = 97.8%, duplicated = 0.9%, fragmented = 0.3%, missing = 1.0%). A total of 98% of the assembled genome was placed into 31 chromosomes, which displayed large-scale synteny with other well-characterized lepidopteran genomes. The E. editha genome, annotation, and functional descriptions now fill a missing gap for one of the leading field-based ecological model systems in North America.