Under the neutral theory, genetic diversity is expected to increase with population size. While comparative analyses have consistently failed to find strong relationships between census population size and genetic diversity, a recent study across animals identified a strong correlation between propagule size and genetic diversity, suggesting that r-strategists that produce many small offspring, have greater long-term population sizes. Here we compare genome-wide genetic diversity across 38 species of European butterflies (Papilionoidea), a group that shows little variation in reproductive strategy. We show that genetic diversity across butterflies varies over an order of magnitude and that this variation cannot be explained by differences in current abundance, propagule size, host or geographic range. Instead, neutral genetic diversity is negatively correlated with body size and positively with the length of the genetic map. This suggests that genetic diversity is determined both by differences in long-term population size and the effect of selection on linked sites.

Chromosomes are a central unit of genome organisation. One tenth of all described species on Earth are Lepidoptera, butterflies and moths, and these generally possess 31 holocentric chromosomes. However, a subset of lepidopteran species display dramatic variation in chromosome counts. By analysing 210 chromosomally-complete lepidopteran genomes, the largest analysis of eukaryotic chromosomal-level reference genomes to date, we show that the diverse karyotypes of extant species are derived from 32 ancestral linkage groups, which we term Merian elements. Merian elements have remained largely intact across 250 million years of evolution and diversification. Against this stable background, we identify eight independent lineages that have evaded constraint and undergone extensive reorganisation - either by numerous fissions or a combination of fusion and fission events. Outside these lineages, fusions are rare and fissions are rarer still. Fusions tend to involve small, repeat-rich Merian elements and/or the Z chromosome. Together, our results reveal the constraints on genome architecture in Lepidoptera and enable a deeper understanding of the importance of chromosomal rearrangements in shaping the evolution of eukaryotic genomes.

Under the neutral theory genetic diversity is expected to be a simple function of population size. However, comparative studies have consistently failed to find any strong correlation between measures of census population size and genetic diversity. Instead, a recent comparative study across several animal phyla identified propagule size as the strongest predictor of genetic diversity, suggesting that r-strategists that produce many offspring but invest little in each, have greater long-term effective population sizes. We present a comparison of genome-wide levels of genetic diversity across 38 species of European butterflies (Papilionoidea). We show that across butterflies, genetic diversity varies over an order of magnitude and that this variation cannot be explained by differences in abundance, fecundity, host plant use or geographic range. Instead, we find that genetic diversity is negatively correlated with body size and positively with the length of the genetic map. This suggests that variation in genetic diversity is determined both by fluctuation in N e and the effect of selection on linked neutral sites.

Chromosome rearrangements are thought to promote reproductive isolation between incipient species. However, it is unclear how often, and under what conditions, fission and fusion rearrangements act as barriers to gene flow. Here we investigate speciation between two largely sympatric fritillary butterflies, Brenthis daphne and B. ino. We use a composite likelihood approach to infer the demographic history of these species from whole genome sequence data. We then compare chromosome-level genome assemblies of individuals from each species and identify a total of nine chromosome fissions and fusions. Finally, we fit a demographic model where effective population sizes and effective migration rate vary across the genome, allowing us to quantify the effects of chromosome rearrangements on reproductive isolation. We show that chromosomes involved in rearrangements experienced less effective migration since the onset of species divergence and that genomic regions near rearrangement points have a further reduction in effective migration rate. Our results suggest that the evolution of multiple rearrangements in the B. daphne and B. ino populations, including alternative fusions of the same chromosomes, have resulted in a reduction in gene flow. While fission and fusion of chromosomes are unlikely to be the only processes that have led to speciation between these butterflies, this study shows that these rearrangements can directly promote reproductive isolation and may be involved in speciation when karyotypes evolve quickly.

The lesser marbled fritillary, Brenthis ino (Rottemburg, 1775), is a species of Palearctic butterfly. Male B. ino individuals have been reported to have between 12 and 14 pairs of chromosomes, a much reduced chromosome number than is typical in butterflies. Here we present a chromosome-level genome assembly for B. ino, as well as gene and transposable element annotations. The assembly is 411.8 Mb in span with contig and scaffold N50s of 9.6 and 29.5 Mb respectively. We also show evidence that the male individual from which we generated HiC data was heterozygous for a neo-Z chromosome, consistent with inheriting 14 chromosomes from one parent and 13 from the other. This genome assembly will be a valuable resource for studying chromosome evolution in Lepidoptera, as well as for comparative and population genomics more generally.

Long-distance migration requires adaptations in a suite of behavioral, physiological and morphological traits. However, almost nothing is known about the genetic basis of these adaptations. The willow warbler Phylloscopus trochilus occurs in Europe with a northern and southern subspecies that show drastically different migration routes and wintering areas. Previous studies have demonstrated that the subspecies are genetically extremely similar except for three divergent chromosome regions, of which two are associated with the differences in migratory phenotypes and one is associated with an environmental gradient. Here we use a combination of long-read sequencing, linked-read sequencing and optical mapping to construct more complete and contiguous assemblies for both of the subspecies. We find evidence for inversions in each of the three divergent regions, which range from 0.4 to 13 Mb in size, and that breakpoints are associated with tandem repeat arrays or segmental duplications. The divergence times between inverted and non-inverted haplotypes are similar across the regions (~1.2 Myrs), which is compatible with a scenario where the inversions arose in either of two allopatric populations that subsequently hybridized. The improved genome assemblies and annotation also allowed us to detect additional functional differences in the divergent regions that provide candidate genes for migration and local adaptations to environmental gradients.

The lesser marbled fritillary, Brenthis ino (Rottemburg, 1775), is a species of Palearctic butterfly. Male B. ino individuals have been reported to have between 12 and 14 pairs of chromosomes, a much reduced chromosome number than is typical in butterflies. Here we present a chromosome-level genome assembly for B. ino, as well as gene and transposable element annotations. The assembly is 411.8 Mb in length with a contig N50 of 9.6 Mb and a scaffold N50 of 29.5 Mb. We also show evidence that the male individual from which we generated HiC data was heterozygous for a neo-Z chromosome, consistent with inheriting 14 chromosomes from one parent and 13 from the other. This genome assembly will be a valuable resource for studying chromosome evolution in Lepidoptera, as well as for comparative and population genomics more generally.

Structural rearrangements have been shown to be important in local adaptation and speciation, but have been difficult to reliably identify and characterize in non-model species. Here we combine long reads, linked reads and optical mapping to characterize three divergent chromosome regions in the willow warbler Phylloscopus trochilus, of which two are associated with differences in migration and one with an environmental gradient. We show that there are inversions (0.4–13 Mb) in each of the regions and that the divergence times between inverted and non-inverted haplotypes are similar across the regions (~1.2 Myrs), which is compatible with a scenario where inversions arose in either of two allopatric populations that subsequently hybridized. The improved genomes allow us to detect additional functional differences in the divergent regions, providing candidate genes for migration and adaptations to environmental gradients.