To date, the use of CRISPR/Cas9 technology in ecological-model species for validating genotype to phenotype connections has focused primarily on visual phenotypes using G0 mutations, coupled with analyses of resulting mosaic phenotypes. However, studies of physiological phenotypes necessitate germline mutations in order to assess non-visible phenotypic effects, and thus, dedicated efforts to develop efficient germline mutations in ecological model species are needed. Here, we applied the CRISPR/Cas9 technology to an ecological model species, the speckled wood butterfly (Pararge aegeria). We targeted yellow-y, which is required for the production of black melanin, as yellow-y loss of function (LOF) mutations are not lethal and easy to phenotype, affording efficient assessment of G0 and germline mutations. To explore what factors may affect the efficiency of transformation, we employed four alternative treatments, including variation in sgRNAs and their concentrations. Color changes in the head capsule of first larval instars, as well as adult wing color, were used as indicators of successful knockouts. Individuals with wings that were at least 50% transformed were mated, with their G1 offspring assessed for the presence of germline mutations. Our CRISPR/Cas9 technique was highly efficient at generating LOF mutations in yellow-y. Across all treatments, nearly 80% of adults exhibited mosaic LOF phenotypes, of which nearly 30% appeared to have 100% LOF phenotypes. Crosses between adults exhibiting at least 50% LOF phenotypes resulted in fully transformed offspring, revealing a high incidence of germline LOF mutations in yellow-y. We provide a detailed protocol on how to obtain high germline LOF mutation efficiency in order to advance the study of genotype–phenotype connections for non-visible physiological traits across natural populations of this and other model ecological species.

Parasitoid wasps are major causes of mortality of many species, making host immune defences a common target of adaptive evolution, though such targets outside model species are poorly understood. In this study, we used two tests of positive selection to compare across three closely related Galerucella leaf beetles that show substantial differences in their phenotypic response to the shared parasitoid wasp Asecodes parviclava, their main natural enemy. Using a codon-based test, which detects excess amino acid fixations per locus along each species’ lineage, we found more evidence of positive selection on parasitoid-relevant immune genes in the species with the strongest immunocompetence (G. pusilla) compared with the species having weaker immunocompetence (G. tenella and G. calmariensis). Moreover, genes coding for the early phases in the immune response cascade were predominantly among the positively selected immune genes, providing targets for future functional genomic study to pin-point connections between genotypic and phenotypic differences in defences towards a parasitoid wasp. In contrast, genome-wide analyses of the haplotype frequency spectrum, which quantify selection over recent evolutionary time scales, revealed similar signatures of positive selection on immune genes across species. These results advance the field of host-parasitoid dynamics by providing novel insights into the tempo and mode of insect host evolutionary dynamics, and offering a framework for making genotype to phenotype connections for immunocompetence phenotypes.

Sex-limited morphs can provide profound insights into the evolution and genomic architecture of complex phenotypes. Inter-sexual mimicry is one particular type of sex-limited polymorphism in which a novel morph resembles the opposite sex. While inter-sexual mimics are known in both sexes and a diverse range of animals, their evolutionary origin is poorly understood. Here, we investigated the genomic basis of female-limited morphs and male mimicry in the common bluetail damselfly. Differential gene expression between morphs has been documented in damselflies, but no causal locus has been previously identified. We found that male mimicry originated in an ancestrally sexually dimorphic lineage in association with multiple structural changes, probably driven by transposable element activity. These changes resulted in similar to 900 kb of novel genomic content that is partly shared by male mimics in a close relative, indicating that male mimicry is a trans-species polymorphism. More recently, a third morph originated following the translocation of part of the male-mimicry sequence into a genomic position similar to 3.5 mb apart. We provide evidence of balancing selection maintaining male mimicry, in line with previous field population studies. Our results underscore how structural variants affecting a handful of potentially regulatory genes and morph-specific genes can give rise to novel and complex phenotypic polymorphisms.

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. 

Understanding how novel traits evolve is a central question in evolutionary biology. However, unraveling the complex genetic and developmental mechanisms underlying trait innovation can be challenging, especially when the trait evolved a long time ago. One approach to this complexity is to leverage natural polymorphisms, revealing variation in the expression or function of novel traits. Polymorphisms can provide insights into the origin, maintenance, and diversification of trait innovation, and the evolutionary forces and constraints shaping them. Sex-limited polymorphisms, a special class of polymorphism in which only one sex exhibits variation in the trait, can be particularly informative because they allow for the investigation of not only the genetic basis and evolutionary history of novel traits, but also how differences between sexes (sexual dimorphisms) are regulated and evolve.

In my thesis, I utilize naturally occurring female-limited color polymorphisms to answer questions about the evolutionary origin, mechanism, and maintenance of wing coloration and patterning variation in butterflies.  Butterfly wing coloration and patterning are not only striking examples of morphological diversity but also critical to survival and fitness. While butterfly wing patterning and coloration have inspired evolutionary thinking  for over a century, they have recently become a model system for Evo-Devo research. As easily visually assessed traits, butterfly wings have proven perfect candidates for more recent advances in Evo-Devo, acting as a template for understanding the function, recruitment, and evolution of gene regulatory networks (GRNs) generating complex phenotypes. 

In Paper I, I reconstruct the evolutionary history of Alba, a genetically determined female-limited alternative life history strategy, found in over one third of species in the genus Colias (Pieridae). In this polymorphism, some Colias females redirect resources from wing coloration to reproductive development, resulting in white rather than orange wings. I show this polymorphism evolved once in the Colias phylogeny through a transposable element insertion and has persisted for over a million years through balancing selection and introgression. In Paper II, I dissect the pteridine biosynthesis pathway, a pigment that Pierid butterflies, including Colias, use to color their wings. I highlight not only the extent of pteridine use by Pierid butterflies, but also evidence suggesting novel evolution for core components of the biosynthesis pathway. In Paper III, I investigate the genetic basis of a regionally isolated sexual dimorphism in Pieris napi, called adalvinda. Rather than the creamy white wings seen in the rest of the range, females in northern Scandinavian populations have highly melanized, almost dark brown wings. Similarly to Paper I, I in Paper III identify a transposable element insertion, but this time upstream of the gene cortex associated with female wing melanization. This finding contributes to a growing body of literature linking cortex with butterfly wing melanization, while emphasizing the potential role transposable elements may play in the evolution of novel – and especially female-limited or sexually dimorphic – traits. Lastly, in Paper IV, I present a new reference genome for the Edith's Checkerspot butterfly, offering an important resource for future functional genomic and conservation analyses, and demonstrating an efficient framework for developing genetic resources for non-model systems. 

In summary, my thesis demonstrates the powerful potential of utilizing naturally occurring polymorphisms or induced mutations to study the evolution of novel traits.

Mating cues evolve rapidly and can contribute to species formation and maintenance. However, little is known about how sexual signals diverge and how this variation integrates with other barrier loci to shape the genomic landscape of reproductive isolation. Here, we elucidate the genetic basis of ultraviolet (UV) iridescence, a courtship signal that differentiates the males of Colias eurytheme butterflies from a sister species, allowing females to avoid costly heterospecific matings. Anthropogenic range expansion of the two incipient species established a large zone of secondary contact across the eastern United States with strong signatures of genomic admixtures spanning all autosomes. In contrast, Z chromosomes are highly differentiated between the two species, supporting a disproportionate role of sex chromosomes in speciation known as the large-X (or large-Z) effect. Within this chromosome-wide reproductive barrier, linkage mapping indicates that cis-regulatory variation of bric a brac (bab) underlies the male UV-iridescence polymorphism between the two species. Bab is expressed in all non-UV scales, and butterflies of either species or sex acquire widespread ectopic iridescence following its CRISPR knockout, demonstrating that Bab functions as a suppressor of UV-scale differentiation that potentiates mating cue divergence. These results highlight how a genetic switch can regulate a premating signal and integrate with other reproductive barriers during intermediate phases of speciation.

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. 

Coevolutionary interactions are responsible for much of the Earth’s biodiversity, with key innovations driving speciation bursts on both sides of the interaction. One persistent question is whether macroevolutionary traits identified as key innovations accurately predict functional performance and selection dynamics within species, as this necessitates characterizing their function, investigating their fitness consequences, and exploring the selection dynamics acting upon them. Here, we used CRISPR-Cas9 mediating nonhomologous end joining (NHEJ) in the butterfly species Pieris brassicae to knock out and directly assess the function and fitness impacts of nitrile specifier protein (NSP) and major allergen (MA). These are two closely related genes that facilitate glucosinolate (GSL) detoxification capacity, which is a key innovation in mustard feeding Pierinae butterflies. We find NSP and MA are both required for survival on plants containing GSLs, with expression differences arising in response to variable GSL profiles, concordant with detoxification performance. Importantly, this concordance was only observed when using natural host plants, likely reflecting the complexity of how these enzymes interact with natural plant variation in GSLs and myrosinases. Finally, signatures of positive selection for NSP and MA were detected across Pieris species, consistent with these genes’ importance in recent coevolutionary interactions. Thus, the war between these butterflies and their host plants involves more than the mere presence of chemical defenses and detoxification mechanisms, as their regulation and activation represent key components of complex interactions. We find that inclusion of these dynamics, in ecologically relevant assays, is necessary for coevolutionary insights in this system and likely others.

Conservation genomics has made dramatic improvements over the past decade, leveraging the power of genomes to infer diverse parameters central to conservation management questions. However, much of this effort has focused upon vertebrate species, despite insects providing similar flagship status with the added benefit of smaller genomes, shorter generation times and extensive historical collections in museums. Here we present the genome of the Apollo butterfly (Parnassius apollo, Papilionidae), an iconic endangered butterfly, which like many species in this genus, needs conservation genomic attention yet lacks a genome. Using 68.7 Gb of long -read data (N50 = 15.2 kb) we assembled a 1.4 Gb genome for the Apollo butterfly, making this the largest sequenced Lepidopteran genome to date. The assembly was highly contiguous (N50 = 7.1 Mb) and complete (97% of Lepidopteran BUSCOs were single-copy and complete) and consisted of 1,707 contigs. Using RNAseq data and Arthropoda proteins, we annotated 28.3K genes. Alignment with the closest-related chromosome-level assembly, Papilio bianor, reveals a highly conserved chromosomal organization, albeit genome size is highly expanded in the Apollo butterfly, due primarily to a dramatic increase in repetitive element content. Using this alignment for superscaffolding places the P. apollo genome in to 31 chromosomal scaffolds, and together with our functional annotation, provides an essential resource for advancing conservation genomics in a flagship species for insect conservation.

Tradeoffs affect resource allocation during development and result in fitness consequences that drive the evolution of life history strategies. Yet despite their importance, we know little about the mechanisms underlying life history tradeoffs. Many species of Colias butterflies exhibit an alternative life history strategy (ALHS) where females divert resources from wing pigment synthesis to reproductive and somatic development. Due to this reallocation, a wing color polymorphism is associated with the ALHS: either yellow/orange or white. Here we map the locus associated with this ALHS in Colias crocea to a transposable element insertion located downstream of the Colias homolog of BarH-1, a homeobox transcription factor. Using CRISPR/Cas9 gene editing, antibody staining, and electron microscopy we find white-specific expression of BarH-1 suppresses the formation of pigment granules in wing scales and gives rise to white wing color. Lipid and transcriptome analyses reveal physiological differences associated with the ALHS. Together, these findings characterize a mechanism for a female-limited ALHS.