Comparative Analyses of Four Reference Genomes Reveal Exceptional Diversity and Weak Linked Selection in the Yellow Monkeyflower (Mimulus guttatus) Complex

Yellow monkeyflowers (Mimulus guttatus complex, Phrymaceae) are a powerful system for studying ecological adaptation, reproductive variation, and genome evolution. To initiate pan-genomics in this group, we present four chromosome-scale assemblies and annotations of accessions spanning a broad evolutionary spectrum: two from a single M. guttatus population, one from the closely related selfing species M. nasutus, and one from a more divergent species M. tilingii. All assemblies are highly complete and resolve centromeric and repetitive regions. Comparative analyses reveal such extensive structural variation in repeat-rich, gene-poor regions that large portions of the genome are unalignable across accessions. As a result, this Mimulus pan-genome is primarily informative in genic regions, underscoring limitations of resequencing approaches in such polymorphic taxa. We document gene presence–absence, investigate the recombination landscape using high-resolution linkage data, and quantify nucleotide diversity. Surprisingly, pairwise differences at fourfold synonymous sites are exceptionally high—even in regions of very low recombination—reaching ~3.2% within a single M. guttatus population, ~7% within the interfertile M. guttatus species complex (approximately equal to SNP divergence between great apes and Old World monkeys), and ~7.4% between that complex and the reproductively isolated M. tilingii. Genome-wide patterns of nucleotide variation show little evidence of linked selection, and instead suggest that the concentration of genes (and likely selected sites) in high-recombination regions may buffer diversity loss. These assemblies, annotations, and comparative analyses provide a robust genomic foundation for Mimulus research and offer new insights into the interplay of recombination, structural variation, and molecular evolution in highly diverse plant genomes.