Examining differences among recently diverged populations can provide insight into the traits and evolutionary mechanisms that drive or maintain divergence. The genus Sturnella includes 2 recently diverged species, Sturnella magna (Eastern Meadowlark) and S. neglecta (Western Meadowlark), the former of which has a complex of subspecies distributed across the Americas. Of the S. magna subspecies that occur in the United States, S. m. lilianae is the only one with a disjunct range, occurring in the southwestern United States and central Mexico. It also has markedly different song patterns than all other S. magna subspecies. In order to assess population differentiation, we performed whole-genome sequencing of 35 birds and analyzed song characteristics from 85 birds. Songs from each species and S. m. lilianae were diagnosable using linear discriminant function analysis and support divergence in song between all taxa. Phylogenetic analysis and admixture proportions support 3 distinct clades within North American meadowlarks, and tests of introgression failed to detect a significant signal. Overall, our results indicate that S. m. lilianae exhibits high levels of genetic and vocal differentiation from both S. magna and S. neglecta, with no evidence of introgression between any group, and forms a distinct evolutionary lineage. We thus recommend the elevation of S. m. lilianae to species status.
• Understanding species boundaries helps inform policy.
• Meadowlarks are currently classified as two species, but recent research suggests they may be three species.
• A population of Eastern Meadowlarks in the southwestern United States and northern Mexico called Lilian's Meadowlark have lighter plumage color and sing different songs than the rest of Eastern Meadowlarks.
• Using DNA and birdsong, we compared populations of Eastern, Western, and Lilian's meadowlarks to find how they were related to one another.
• Eastern, Western, and Lilian's meadowlarks all have moderate but equal amounts of differentiation from one another, suggesting Lilian's Meadowlarks is a full species.