The evolution and diversification of oakleaf butterflies.

Oakleaf butterflies in the genus Kallima have a polymorphic wing phenotype, enabling these insects to masquerade as dead leaves. This iconic example of protective resemblance provides an interesting evolutionary paradigm that can be employed to study biodiversity. We integrated multi-omic data analyses and functional validation to infer the evolutionary history of Kallima species and investigate the genetic basis of their variable leaf wing patterns. We find that Kallima butterflies diversified in the eastern Himalayas and dispersed to East and Southeast Asia. Moreover, we find that leaf wing polymorphism is controlled by the wing patterning gene cortex, which has been maintained in Kallima by long-term balancing selection. Our results provide macroevolutionary and microevolutionary insights into a model species originating from a mountain ecosystem.

Chromatin accessibility profiling provides insights into larval cuticle color and adult longevity in butterflies.

Butterflies are diverse in virtually all aspects of their ontogeny, including morphology, life history, and behavior. However, the developmental regulatory mechanisms underlying the important phenotypic traits of butterflies at different developmental stages remain unknown. Here, we investigated the developmental regulatory profiles of butterflies based on transposase accessible chromatin sequencing (ATAC-seq) at three developmental stages in two representative species ( Papilio xuthus and Kallima inachus). Results indicated that 15%-47% of open chromatin peaks appeared in associated genes located 3 kb upstream (i.e., promoter region) of their transcription start site (TSS). Comparative analysis of the different developmental stages indicated that chromatin accessibility is a dynamic process and associated genes with differentially accessible (DA) peaks show functions corresponding to their phenotypic traits. Interestingly, the black color pattern in P. xuthus 4th instar larvae may be attributed to promoter peak-related genes involved in the melanogenesis pathway. Furthermore, many longevity genes in 5th instar larvae and pupae showed open peaks 3 kb upstream of their TSS, which may contribute to the overwintering diapause observed in K. inachus adults. Combined with RNA-seq analysis, our data demonstrated that several genes enriched in the melanogenesis and longevity pathways also exhibit higher expression, confirming that the expression of genes may be closely related to their phenotypic traits. This study offers new insights into larval cuticle color and adult longevity in butterflies and provides a resource for investigating the developmental regulatory mechanisms underlying butterfly ontogeny.

Mimicking Nature's Butterflies: Electrochromic Devices with Dual-Sided Differential Colorations.

Some butterfly species such as the orange oakleaf (Kallima inachus) have strikingly different colors on the dorsal (front) sides of their wings compared to those on the ventral (back) sides of their wings, which helps camouflage the butterflies from predators and attract potential mates. However, few human-made materials, devices, and technologies can mimic such differential coloring for a long time. Here, a new type of Janus-structured two-sided electrochromic device is developed that, upon application of different voltages, exhibits a coloration state on one side that is distinctly different from that on the other side. This is achieved by inserting an optically thin (4-8 nm) metallic layer with a complex refractive index, such as a layer composed of tungsten, titanium, copper or silver, into typical electrochromic structures.

Chromosome-level reference genome assembly and gene editing of the dead-leaf butterfly Kallima inachus.

The leaf resemblance of Kallima (Nymphalidae) butterflies is an important ecological adaptive mechanism that increases their survival. However, the genetic mechanism underlying ecological adaptation remains unclear owing to a dearth of genomic information. Here, we determined the karyotype (n = 31) of the dead-leaf butterfly Kallima inachus, and generated a high-quality, chromosome-level assembly (568.92 Mb; contig N50: 19.20 Mb). We also identified candidate Z and W chromosomes. To our knowledge, this is the first study to report on these aspects of this species. In the assembled genome, 15,309 protein-coding genes and 49.86% repeat elements were annotated. Phylogenetic analysis showed that K. inachus diverged from Melitaea cinxia (no leaf resemblance), both of which are in Nymphalinae, around 40 million years ago. Demographic analysis indicated that the effective population size of K. inachus decreased during the last interglacial period in the Pleistocene. The wings of adults with the pigmentary gene ebony knocked out using CRISPR/Cas9 showed phenotypes in which the orange dorsal region and entire ventral surface darkened, suggesting its vital role in the ecological adaption of dead-leaf butterflies. Our results provide important genome resources for investigating the genetic mechanism underlying protective resemblance in dead-leaf butterflies and insights into the molecular basis of protective coloration.