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Species-specific sexual traits facilitate species-assortative mating by reducing mating across species and reducing hybrid sexual attractiveness. For learned sexual traits, such as song in oscine birds, species distinctiveness can be eroded when species co-occur. Transcriptional regulatory divergence in brain regions involved in sensory learning is hypothesized to maintain species distinctiveness, but relatively few studies have compared gene expression in relevant brain regions between closely related species. Species differences in song are an important premating reproductive barrier between the collared (Ficedula albicollis) and pied flycatcher (F. hypoleuca). Here, we compare brain gene expression in adult males from each species and their naturally occurring F1 hybrids. We report overall conserved expression across species in a portion of the brain containing regions and nuclei known to be involved in song responses and learning. Further, among those genes that were differentially expressed between species, we find largely intermediate expression in hybrids. A single gene, SYT4 (synaptotagmin 4), known to be singing-associated, both was differentially expressed and has a putative upstream transcriptional regulatory factor containing fixed differences between the 2 species. Although a finer-scale investigation limited to song-specific regions may reveal further species differences, our findings provide insight into regulatory divergence in the brain between closely related species.

The painted lady butterfly Vanessa cardui is renowned for its virtually cosmopolitan distribution and the remarkable long-distance migrations as part of its annual, multigenerational migratory cycle. In winter, V. cardui individuals inhabit breeding grounds north and south of the Sahara, suggesting distinct migratory behaviors within the species as individuals migrate southward from Europe in the autumn. However, the evolutionary and ecological factors shaping these differences in migratory behavior remain largely unexplored. Here, we performed whole-genome resequencing and analyzed the hydrogen and strontium isotopes of 40 V. cardui individuals simultaneously collected in the autumn from regions both north and south of the Sahara. Our investigation revealed two main migratory groups: (i) short-distance migrants, journeying from temperate Europe to the circum-Mediterranean region and (ii) long-distance migrants, originating from Europe, crossing the Mediterranean Sea and Sahara, and reaching West Africa, covering up to over 4,000 km. Despite these stark differences in migration distance, a genome-wide analysis revealed that short- and long-distance migrants belong to a single intercontinental panmictic population extending from northern Europe to sub-Saharan Africa. Contrary to common biogeographic patterns, the Sahara is not a catalyst for population structuring in this species. No significant genetic differentiation or signs of adaptation and selection were observed between the two migratory phenotypes. Nonetheless, two individuals, who were early arrivals to West Africa covering longer migration distances, exhibited some genetic differentiation. The lack of genetic structure between short- and long-distance migrants suggests that migration distance in V. cardui is a plastic response to environmental conditions.

A new species can form through hybridization between species. Hybrid speciation in animals has been intensely debated, partly because hard evidence for the process has been difficult to obtain. Here, we report the discovery of a European hybrid butterfly lineage, a finding that can be considered surprising given the intense and long-term study of European butterflies. The lineage we describe is mainly inhabiting an island in the Baltic Sea and was previously designated as a subspecies (horkei) of one of the parental species (Aricia artaxerxes). By analyzing whole-genome resequencing data and developing a novel cluster analysis based on historical recombination events (Fisher junctions), we determine that horkei originated by hybridization between the nonsister species A. artaxerxes and A. agestis. This hybridization event occurred approximately 54,000 years ago, predating the last glaciation of the current distribution range. Horkei must therefore have persisted long enough to be able to colonize its current range, despite that this area lies between the current distributions of the parental species. The hybrid origin, the maintenance of genomic integrity across times of dramatic climate change, and the expression of a combination of parental traits suggest that horkei could be in the process of hybrid speciation.

The clouded apollo (Parnassius mnemosyne) is a palearctic butterfly distributed over a large part of western Eurasia, but population declines and fragmentation have been observed in many parts of the range. The development of genomic tools can help to shed light on the genetic consequences of the decline and to make informed decisions about direct conservation actions. Here, we present a high-contiguity, chromosome-level genome assembly of a female clouded apollo butterfly and provide detailed annotations of genes and transposable elements. We find that the large genome (1.5 Gb) of the clouded apollo is extraordinarily repeat rich (73%). Despite that, the combination of sequencing techniques allowed us to assemble all chromosomes (n_c = 29) to a high degree of completeness. The annotation resulted in a relatively high number of protein-coding genes (22,854) compared with other Lepidoptera, of which a large proportion (21,635) could be assigned functions based on homology with other species. A comparative analysis indicates that overall genome structure has been largely conserved, both within the genus and compared with the ancestral lepidopteran karyotype. The high-quality genome assembly and detailed annotation presented here will constitute an important tool for forthcoming efforts aimed at understanding the genetic consequences of fragmentation and decline, as well as for assessments of genetic diversity, population structure, inbreeding, and genetic load in the clouded apollo butterfly.

Species frequently differ in the number and structure of chromosomes they harbor, but individuals that are heterozygous for chromosomal rearrangements may suffer from reduced fitness. Chromosomal rearrangements like fissions and fusions can hence serve as a mechanism for speciation between incipient lineages, but their evolution poses a paradox. How can rearrangements get fixed between populations if heterozygotes have reduced fitness? One solution is that this process predominantly occurs in small and isolated populations, where genetic drift can override natural selection. However, fixation is also more likely if a novel rearrangement is favored by a transmission bias, such as meiotic drive. Here, we investigate chromosomal transmission distortion in hybrids between two wood white (Leptidea sinapis) butterfly populations with extensive karyotype differences. Using data from two different crossing experiments, we uncover that there is a transmission bias favoring the ancestral chromosomal state for derived fusions, a result that shows that chromosome fusions actually can fix in populations despite being counteracted by meiotic drive. This means that meiotic drive not only can promote runaway chromosome number evolution and speciation, but also that it can be a conservative force acting against karyotypic change and the evolution of reproductive isolation. Based on our results, we suggest a mechanistic model for why chromosome fusion mutations may be opposed by meiotic drive and discuss factors contributing to karyotype evolution in Lepidoptera.

The faster-Z/X hypothesis predicts that sex-linked genes should diverge faster than autosomal genes. However, studies across different lineages have shown mixed support for this effect. So far, most analyses have focused on old and well-differentiated sex chromosomes, but less is known about the divergence of more recently acquired neo-sex chromosomes. In Lepidoptera (moths and butterflies), Z-autosome fusions are frequent, but the evolutionary dynamics of neo-Z chromosomes have not been explored in detail. Here, we analyzed the faster-Z effect in Leptidea sinapis, a butterfly with three Z chromosomes. We show that the neo-Z chromosomes have been acquired stepwise, resulting in strata of differentiation and masculinization. While all Z chromosomes showed evidence of the faster-Z effect, selection for genes on the youngest neo-Z chromosome (Z3) appears to have been hampered by a largely intact, homologous neo-W chromosome. However, the intermediately aged neo-Z chromosome (Z2), which lacks W gametologs, showed fewer evolutionary constraints, resulting in particularly fast evolution. Our results therefore support that neo-sex chromosomes can constitute temporary hot-spots of adaptation and divergence. The underlying dynamics are likely causally linked to shifts in selective constraints, evolution of gene expression, and degeneration of W-linked gametologs which gradually expose Z-linked genes to selection.

Coding sequence evolution is influenced by both natural selection and neutral evolutionary forces. In many species, the effects of mutation bias, codon usage and GC-biased gene conversion (gBGC) on gene sequence evolution have not been detailed. Quantification of how these forces shape substitution patterns is therefore necessary to understand the strength and direction of natural selection. Here, we used comparative genomics to investigate the association between base composition and codon usage bias on gene sequence evolution in butterflies and moths (Lepidoptera), including an in-depth analysis of underlying patterns and processes in one species, Leptidea sinapis. The data revealed significant G/C to A/T substitution bias at third codon position with some variation in the strength among different butterfly lineages. However, the substitution bias was lower than expected from previously estimated mutation rate ratios, partly due to the influence of gBGC. We found that A/T-ending codons were overrepresented in most species and there was a negative association between the magnitude of codon usage bias and GC-content in third codon positions. In contrast, the tRNA-gene population in L. sinapis showed higher GC-content at third codon positions compared to coding sequences in general and less overrepresentation of A/T-ending codons. There was an inverse relationship between synonymous substitutions and codon usage bias indicating selection on synonymous sites. We conclude that the evolutionary rate in Lepidoptera is affected by a complex interaction between underlying G/C -> A/T mutation bias and partly counteracting fixation biases, predominantly conferred by overall purifying selection, gBGC and selection on codon usage.

Seasonal environmental fluctuations provide formidable challenges for living organisms, especially small ectotherms such as butterflies. A common strategy to cope with harsh environments is to enter diapause, but some species avoid unsuitable conditions by migrating. Despite a growing understanding of migration in the life cycles of some butterfly species, it remains unknown how individuals register and store environmental cues to determine whether and where to migrate. Here, we explored how competition and host plant availability during larval development affect patterns of DNA methylation in the migratory painted lady (Vanessa cardui) butterfly. We identify a set of potentially functional methylome shifts associated with differences in the environment, indicating that DNA methylation is involved in the response to different conditions during larval development. By analysing the transcriptome for the same samples used for methylation profiling, we also uncovered a non-monotonic relationship between gene body methylation and gene expression. Our results provide a starting point for understanding the interplay between DNA methylation and gene expression in butterflies in general and how differences in environmental conditions during development can trigger unique epigenetic marks that might be important for behavioural decisions in the adult stage.

Karyotypes are generally conserved between closely related species and large chromosome rearrangements typically have negative fitness consequences in heterozygotes, potentially driving speciation. In the order Lepidoptera, most investigated species have the ancestral karyotype and gene synteny is often conserved across deep divergence, although examples of extensive genome reshuffling have recently been demonstrated. The genus Leptidea has an unusual level of chromosome variation and rearranged sex chromosomes, but the extent of restructuring across the rest of the genome is so far unknown. To explore the genomes of the wood white (Leptidea) species complex, we generated eight genome assemblies using a combination of 10X linked reads and HiC data, and improved them using linkage maps for two populations of the common wood white (L. sinapis) with distinct karyotypes. Synteny analysis revealed an extensive amount of rearrangements, both compared to the ancestral karyotype and between the Leptidea species, where only one of the three Z chromosomes was conserved across all comparisons. Most restructuring was explained by fissions and fusions, while translocations appear relatively rare. We further detected several examples of segregating rearrangement polymorphisms supporting a highly dynamic genome evolution in this clade. Fusion breakpoints were enriched for LINEs and LTR elements, which suggests that ectopic recombination might be an important driver in the formation of new chromosomes. Our results show that chromosome count alone may conceal the extent of genome restructuring and we propose that the amount of genome evolution in Lepidoptera might still be underestimated due to lack of taxonomic sampling.

Migration is typically associated with risk and uncertainty at the population level, but little is known about its cost–benefit trade-offs at the species level. Migratory insects in particular often exhibit strong demographic fluctuations due to local bottlenecks and outbreaks. Here, we use genomic data to investigate levels of heterozygosity and long-term population size dynamics in migratory insects, as an alternative to classical local and short-term approaches such as regional field monitoring. We analyse whole-genome sequences from 97 Lepidoptera species and show that individuals of migratory species have significantly higher levels of genome-wide heterozygosity, a proxy for effective population size, than do nonmigratory species. Also, we contribute whole-genome data for one of the most emblematic insect migratory species, the painted lady butterfly (Vanessa cardui), sampled across its worldwide distributional range. This species exhibits one of the highest levels of genomic heterozygosity described in Lepidoptera (2.95 ± 0.15%). Coalescent modelling (PSMC) shows historical demographic stability in V. cardui, and high effective population size estimates of 2–20 million individuals 10,000 years ago. The study reveals that the high risks associated with migration and local environmental fluctuations do not seem to decrease overall genetic diversity and demographic stability in migratory Lepidoptera. We propose a “compensatory” demographic model for migratory r-strategist organisms in which local bottlenecks are counterbalanced by reproductive success elsewhere within their typically large distributional ranges. Our findings highlight that the boundaries of populations are substantially different for sedentary and migratory insects, and that, in the latter, local and even regional field monitoring results may not reflect whole population dynamics. Genomic diversity patterns may elucidate key aspects of an insect's migratory nature and population dynamics at large spatiotemporal scales.