Evaluating Probe Design for Phylogenomics Across Taxonomic Scales: First Steps for Applying Ultraconserved Elements in an Understudied Class (Mollusca: Polyplacophora).

Ultraconserved elements (UCEs) have become a powerful tool for phylogenomics, but probe sets optimized for one lineage often perform inconsistently when applied in others. Here, we designed and tested new UCE probe sets derived from both genome and transcriptome data of an understudied molluscan class, Polyplacophora (chitons). In this study, we identified 5730 ultra-conserved elements (UCEs) from available chiton genomes and transcriptomes, and designed a set of 19,080 probes. These probes showed an average efficiency of 55% in the genome and 20% in transcriptomes, significantly outperforming available molluscan probe sets. A coalescence-based phylogenetic tree based on in silico extractions of UCEs from transcriptome and genome data successfully resolved chiton phylogeny at the species level. Relatively shorter flanking regions performed best. Where genome and transcriptome data were available for the same species, they did not always resolve as sister taxa in non-optimized methods; instead, genome- and transcriptome-derived sequences tended to form separate clades. This offers a caution for combining data harvested from published datasets. Quantifying phylogenetic signal at individual UCE loci demonstrates that the dataset retains topological stability across a range of filtering stringencies. This resource provides a foundation for integrating new genomic and transcriptomic datasets and has the potential to enable targeted sequencing of historical museum specimens. More broadly, our study highlights the importance of tailored probe design for phylogenomic studies in understudied lineages and the challenges of combining diverse molecular data types.