Examining differences among recently diverged populations can provide insight into the traits and evolutionary mechanisms that drive or maintain divergence. The genus Sturnella includes two 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 U.S. 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 three 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.

Shallow whole-genome sequence data of 32 meadowlarks (Eastern, Western, and Lilian's) and 3 Bobolinks. Vcf file has been filtered to a minimum mean depth of 2x and maximum mean depth of 14x per variant, minor allele frequency (10%), no missing data, and removed all scaffolds shorter than 5kb. Song data from 85 meadowlarks (Eastern, Western, and Lilian's) includes measurements taken automatically from manually drawn boundary boxes.