Target enrichment museomics of the Asian long-horned beetle and its relatives (Cerambycidae: Anoplophora) reveals two independent origins of life in the cold

Resolving a robust phylogeny of an organismal group is often hindered by the limited availability of samples suitable for genomic or transcriptomic sequencing. Even for lineages of notable importance in evolutionary ecology, our phylogenetic comprehension remains largely unsatisfactory due to the challenges of acquiring samples across the clade. The long-horned beetle genus Anoplophora Hope exemplifies such a group, globally renowned for two invasive pests-the Asian long-horned beetle and citrus long-horned beetle-which have inflicted significant damage to deciduous hardwood forest in North America and Europe. In contrast to the two temperate pests, the remaining 50 species in the genus inhabit subtropical forests of Southeast Asia, where most species are only infrequently encountered. Here, we present the first comprehensive phylogeny of Anoplophora using a PCR-based target enrichment museomics approach. As a case study of employing PCR-generated custom probes, we demonstrate the robustness and cost-effectiveness of this in-house method in successfully acquiring sequence data from historical specimens. Through extensive sampling of Anoplophora using museum specimens, we reveal a non-sister relationship between the two temperate species and provide evidence for addressing taxonomic conundrums. Our biogeographical analyses indicate that the adaptation of the two temperate species occurred independently during the late Pliocene and Pleistocene after the establishment of temperate forests in East Asia in the late Miocene. Our findings highlight the importance of comprehensive phylogenetic inference in understanding the patterns and processes of these beetles' adaptation to temperate forests and lay the groundwork for investigating the genetic mechanism underlying life in the cold.