LAUSR

Phylogeographical studies of the most species‐rich region of the planet—the Amazon basin—have repeatedly uncovered genetically distinctive, allopatric lineages within currently named species, but understanding whether such lineages are reproductively isolated species is challenging. Here we harness the power of genome‐wide data sets together with detailed phylogeographical sampling to both characterize the number of unique lineages and infer levels of reproductive isolation for three parapatric manakin species that make up the genus Pipra. The mitochondrial and nuclear genomes both support six distinctive lineages. The youngest lineages are now highly admixed with each other across major portions of their geographical ranges with one lineage now extinct in a genomically unadmixed state. In contrast, the oldest sets of lineages—dated to 1.4 million years—exhibit narrow hybrid zones. By fitting demographic models to parapatric lineage pairs we found that levels of gene flow and genomic homogenization decline with increasing evolutionary age. Only lineages descending from the basal node at 1.4 million years ago in the genus experience negligible gene flow, possess genomes resistant to homogenization and are separated by narrow hybrid zones. We conclude that a million years or more were required for Pipra manakins to become reproductively isolated. We suggest the six lineages be reclassified as two or three reproductively isolated species. Our unique approach to quantifying reproductive isolation in parapatric lineages could be applied broadly to other phylogeographical studies and would help determine species classification of the plethora of newly identified lineages in the Amazon basin and other regions.