A pangenomic approach reveals the sources of genetic variation fueling the rapid radiation of Capuchino Seedeaters

The search for the genetic basis of phenotypes has primarily focused on single nucleotide polymorphisms, often overlooking structural variants (SVs). SVs can significantly affect gene function, but detecting and characterizing them is challenging, even with long-read sequencing. Moreover, traditional single-reference methods can fail to capture many genetic variants. Using long reads, we generated a Capuchino Seedeater (Sporophila) pangenome, including 16 individuals from 7 species, to investigate how SVs contribute to species and coloration differences. Leveraging this pangenome, we mapped short-read data from 127 individuals, genotyped variants identified in the pangenome graph, and subsequently performed FST scans and genome-wide association studies. Species divergence primarily arises from SNPs and indels (< 50 bp) in non-coding regions of melanin-related genes, as larger SVs rarely overlap with divergence peaks. One exception was a 55 bp deletion near the OCA2 and HERC2 genes, associated with feather pheomelanin content. These findings support the hypothesis that the reshuffling of small regulatory alleles, rather than larger species-specific mutations, accelerated plumage evolution leading to prezygotic isolation in Capuchinos.