The genomics and evolution of inter-sexual mimicry and female-limited polymorphisms in damselflies.

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 ~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 ~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.

Evidence for a single, ancient origin of a genus-wide alternative life history strategy.

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.

Testing hypotheses of a coevolutionary key innovation reveals a complex suite of traits involved in defusing the mustard oil bomb.

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.

A Genome for Edith's Checkerspot Butterfly: An Insect with Complex Host-Adaptive Suites and Rapid Evolutionary Responses to Environmental Changes.

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.

A genetic switch for male UV iridescence in an incipient species pair of sulphur butterflies.

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.

The Genome Assembly and Annotation of the Apollo Butterfly Parnassius apollo, a Flagship Species for Conservation Biology.

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.

Butterfly dichromatism primarily evolved via Darwin's, not Wallace's, model.

Sexual dimorphism is typically thought to result from sexual selection for elaborated male traits, as proposed by Darwin. However, natural selection could reduce expression of elaborated traits in females, as proposed by Wallace. Darwin and Wallace debated the origins of dichromatism in birds and butterflies, and although evidence in birds is roughly equal, if not in favor of Wallace's model, butterflies lack a similar scale of study. Here, we present a large-scale comparative phylogenetic analysis of the evolution of butterfly coloration, using all European non-hesperiid butterfly species (n = 369). We modeled evolutionary changes in coloration for each species and sex along their phylogeny, thereby estimating the rate and direction of evolution in three-dimensional color space using a novel implementation of phylogenetic ridge regression. We show that male coloration evolved faster than female coloration, especially in strongly dichromatic clades, with male contribution to changes in dichromatism roughly twice that of females. These patterns are consistent with a classic Darwinian model of dichromatism via sexual selection on male coloration, suggesting this model was the dominant driver of dichromatism in European butterflies.

Composition of Strawberry Floral Volatiles and their Effects on Behavior of Strawberry Blossom Weevil, Anthonomus rubi.

The strawberry blossom weevil (SBW), Anthonomus rubi, is a major pest in strawberry fields throughout Europe. Traps baited with aggregation pheromone are used for pest monitoring. However, a more effective lure is needed. For a number of pests, it has been shown that the attractiveness of a pheromone can be enhanced by host plant volatiles. The goal of this study was to explore floral volatile blends of different strawberry species (Fragaria x ananassa and Fragaria vesca) to identify compounds that might be used to improve the attractiveness of existing lures for SBW. Floral emissions of F. x a. varieties Sonata, Beltran, Korona, and of F. vesca, were collected by both solid-phase microextraction (SPME) and dynamic headspace sampling on Tenax. Analysis by gas chromatography/mass spectrometry showed the floral volatiles of F. x ananassa. and F. vesca were dominated by aromatic compounds and terpenoids, with 4-methoxybenzaldehyde (p-anisaldehyde) and α-muurolene the major compounds produced by the two species, respectively. Multi-dimensional scaling analyses separated the blends of the two species and explained differences between F. vesca genotypes and, to some degree, variation between F. x ananassa varieties In two-choice behavioral tests, SBW preferred odors of flowering strawberry plants to those of non-flowering plants, but weevils did not discriminate between odors from F. x ananassa and F. vesca flowering plants. Adding blends of six synthetic flower volatiles to non-flowering plants of both species increased the preference of SBW for these over the plants alone. When added individually to non-flowering plants, none of the components increased the preference of SBW, indicating a synergistic effect. However, SBW responded to 1,4-dimethoxybenzene, a major component of volatiles from F. viridis, previously found to synergize the attractiveness of the SBW aggregation pheromone in field studies.

A transposable element insertion is associated with an alternative life history strategy.

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.