Chromosome-scale Juglans genomes focus fungal defense gene divergence in gene presence and absence instead of copy number

Butternut (Juglans cinerea) is a North American hardwood in steep decline, driven largely by an introduced fungal pathogen that causes butternut canker disease. Tolerance exists within the genus, with the Asian Juglans ailantifolia comparatively tolerant and hybridizing readily with J. cinerea, yet the genomic basis of that difference is poorly resolved. Chromosome-scale reference genomes are presented for J. ailantifolia and for a second J. cinerea accession from the species primary range, with independent scaffolding and uniform annotation across all compared assemblies. Gene family evolution was assessed across twelve Juglandaceae genomes, one per species, and a four accession pangenome was built from J. ailantifolia and J. mandshurica representing section Cardiocaryon and two J. cinerea accessions, Brenton and New Brunswick, representing section Trachycaryon. Neither J. cinerea accession is a putative tolerant selection, so their contrast provides a null against which section-level differences are calibrated. Spanning two sections separated by approximately 30 My, the pangenome partitions many tandem arrays by accession instead of by gene, so copy number is interpreted after merging homology nodes representing the same gene. Section-level variation resides primarily in gene presence and absence, where pathogenesis-related proteins, cysteine-rich receptor-like kinases, wall-associated kinases and lectin-domain receptor kinases are over-represented in the lineage-specific complement of both sections, indicating rapid bidirectional turnover. A smaller set of families is asymmetric, with chitinases and chitin-binding proteins and NB-LRR receptors biased toward the tolerant section and dehydrins, pectin-modifying enzymes and the CBF regulon toward the susceptible one. The largest copy number difference in the dataset separates the two conspecific accessions, at a senescence-associated cysteine protease. The two butternuts are further distinguished by their demographic histories and by the functional composition of their accession-specific gene content: the northern New Brunswick accession retains lower heterozygosity, a distinct coalescent trajectory, and enrichment for calcium transport across channels, pumps and homeostasis, for salt stress response regulation, and for raffinose family oligosaccharide biosynthesis, spanning both the perception and the cryoprotective effectors of freezing tolerance. These findings support recognition of the northern population as a cryptic glacial refugium and a conservation priority.