Supplementary material for Klementz et al. (2025) Exploring genome architecture as a source of phylogenetic characters for resolving the apulmonate arachnid polytomy. Molecular Phylogenetics and Evolution.

Chromosome-level genome assemblies are powerful tools for identifying the presence of rare genomic changes that can overcome phylogenetically intractable problems. Chelicerata, the sister group to the remaining arthropods, harbors a soft polytomy at the base of an internal node named Euchelicerata, which is variably resolved across phylogenomic studies. As a result, seven orders, comprising horseshoe crabs and six apulmonate arachnid lineages, exhibit highly unstable placements from one study to the next, typically with maximal nodal support. Here, we analyzed recently released chromosome-level genomes of two of these orders, Opiliones (harvestmen) and Solifugae (camel spiders). We show that both Opiliones and Solifugae exhibit an unduplicated genome condition, as inferred from analysis of gene clusters, microRNAs, and macrosynteny. These results are congruent with phylogenomic studies that have refuted traditional morphological placements of Opiliones and Solifugae as close relatives of orders within Arachnopulmonata, a subset of six arachnid orders that are united by a shared whole genome duplication. Additionally, we examine irreversible chromosome fusion-with-mixing events as potential sources of phylogenetic data. We show that while fusion and mixing events are common in apulmonate arachnids, multiple mixing events support incompatible unrooted tree topologies. These results suggest that fusion and mixing events have evolved convergently in the chelicerate tree of life, particularly for extant lineages with a small number of chromosomes. Overall, our findings demonstrate that broader sampling of chelicerate genomes and establishment of genomic resources for key missing orders are essential to unlocking the potential of rare genomic changes as phylogenetic data sources.