Phylogenomic and comparative genome analysis of the Juglandaceae family reveals black walnut ( Juglans nigra ) as an ancient lineage of the Rhysocaryon section

Accelerated climate change causes heatwaves, droughts, and severe freezes, posing threats to tree regeneration and to global biodiversity. Deeper genomic insights are crucial for breeding, conserving genetic resources, guiding management, and building resilience to these threats. Juglans , a globally distributed genus of timber and nut-producing trees, still has unresolved species relationships; comprehensive genomic data would clarify adaptation to rapid climate change and inform assisted migration strategies. We analyzed 20 Juglans species from North and South America and Asia using whole-genome data, chloroplast genome data, cold-hardiness gene families, and environmental variables related to freezing tolerance. Phylogenomic analyses indicate that black walnuts ( J. nigra , section Rhysocaryon ) may have diverged ~28.7 MYA (late Oligocene). J. nigra reveals higher nucleotide diversity with more genes than other Juglans , whereas J. hindsii clusters with Central/South American species, consistent with a more recent divergence within Rhysocaryon . As expected, Juglans species originating from mild-winter regions are distinct from those in cold-climate regions in genome-wide and cold-hardiness gene analyses, with introgression occurring mainly from temperate into tropical lineages. Juglans species from milder or warmer winters show CBF gene deletions, whereas cold-adapted species lack such gene deletions. These results support J. nigra as an ancient lineage within Rhysocaryon, consistent with subsequent colonization of Central/South America and longer evolutionary histories. Genome analyses further suggest Rhysocaryon is ancestral to Cardicaryon, Trachycaryon, and Dioscaryon. These findings illuminate the demographic history of the Juglans , particularly Rhysocaryon, and have direct implications for gene conservation, breeding, and assisted migration.