Genome structure-based Juglandaceae phylogenies contradict alignment-based phylogenies and substitution rates vary with DNA repair genes.

In lineages of allopolyploid origin, sets of homoeologous chromosomes may coexist that differ in gene content and syntenic structure. Presence or absence of genes and microsynteny along chromosomal blocks can serve to differentiate subgenomes and to infer phylogenies. We here apply genome-structural data to infer relationships in an ancient allopolyploid lineage, the walnut family (Juglandaceae), by using seven chromosome-level genomes, two of them newly assembled. Microsynteny and gene-content analyses yield identical topologies that place Platycarya with Engelhardia as did a 1980s morphological-cladistic study. DNA-alignment-based topologies here and in numerous earlier studies instead group Platycarya with Carya and Juglans, perhaps misled by past hybridization. All available data support a hybrid origin of Juglandaceae from extinct or unsampled progenitors nested within, or sister to, Myricaceae. Rhoiptelea chiliantha, sister to all other Juglandaceae, contains proportionally more DNA repair genes and appears to evolve at a rate 2.6- to 3.5-times slower than the remaining species.