High-quality reference genomes of swallowtail butterflies provide insights into their coloration evolution.

Swallowtail butterflies (Papilionidae) are a historically significant butterfly group due to their colorful wing patterns, extensive morphological diversity, and phylogenetically important position as a sister group to all other butterflies and have been widely studied regarding ecological adaption, phylogeny, genetics, and evolution. Notably, they contain a unique class of pigments, i.e., papiliochromes, which contribute to their color diversity and various biological functions such as predator avoidance and mate preference. To date, however, the genomic and genetic basis of their color diversity and papiliochrome origin in a phylogenetic and evolutionary context remain largely unknown. Here, we obtained high-quality reference genomes of 11 swallowtail butterfly species covering all tribes of Papilioninae and Parnassiinae using long-read sequencing technology. Combined with previously published butterfly genomes, we obtained robust phylogenetic relationships among tribes, overcoming the challenges of incomplete lineage sorting (ILS) and gene flow. Comprehensive genomic analyses indicated that the evolution of Papilionidae-specific conserved non-exonic elements (PSCNEs) and transcription factor binding sites (TFBSs) of patterning and transporter/cofactor genes, together with the rapid evolution of transporters/cofactors, likely promoted the origin and evolution of papiliochromes. These findings not only provide novel insights into the genomic basis of color diversity, especially papiliochrome origin in swallowtail butterflies, but also provide important data resources for exploring the evolution, ecology, and conservation of butterflies.

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.

Chromosomal-level reference genome of Chinese peacock butterfly (Papilio bianor) based on third-generation DNA sequencing and Hi-C analysis.

BACKGROUND: Papilio bianor Cramer, 1777 (commonly known as the Chinese peacock butterfly) (Insecta, Lepidoptera, Papilionidae) is a widely distributed swallowtail butterfly with a wide number of geographic populations ranging from the southeast of Russia to China, Japan, India, Vietnam, Myanmar, and Thailand. Its wing color consists of both pigmentary colored scales (black, reddish) and structural colored scales (iridescent blue or green dust). A high-quality reference genome of P. bianor is an important foundation for investigating iridescent color evolution, phylogeography, and the evolution of swallowtail butterflies. FINDINGS: We obtained a chromosome-level de novo genome assembly of the highly heterozygous P. bianor using long Pacific Biosciences sequencing reads and high-throughput chromosome conformation capture technology. The final assembly is 421.52 Mb on 30 chromosomes (29 autosomes and 1 Z sex chromosome) with 13.12 Mb scaffold N50. In total, 15,375 protein-coding genes and 233.09 Mb of repetitive sequences were identified. Phylogenetic analyses indicated that P. bianor separated from a common ancestor of swallowtails ∼23.69-36.04 million years ago. Demographic history suggested that the population expansion of this species from the last interglacial period to the last glacial maximum possibly resulted from its decreased natural enemies and its adaptation to climate change during the glacial period. CONCLUSIONS: We present a high-quality chromosome-level reference genome of P. bianor using long-read single-molecule sequencing and Hi-C-based chromatin interaction maps. Our results lay the foundation for exploring the genetic basis of special biological features of P. bianor and also provide a useful data source for comparative genomics and phylogenomics among butterflies and moths.