The saltmarsh sparrow Ammospiza caudacuta and the Nelson’s sparrow A. nelsoni differ in ecological niche, mating behavior, and plumage, but they hybridize where their breeding distributions overlap. In this advanced hybrid zone, past inter-breeding and current back-crossing result in substantial genomic introgression in both directions, although few hybrids are currently produced in most locations. However, because both species are non-territorial and have only brief male-female interactions, it is difficult to determine to what extent assortative mating explains the low frequency of hybrid offspring. Since females often copulate with multiple males, a role of sperm as a post-copulatory pre-zygotic barrier appears plausible. Here we show that sperm length differs between the two species in the hybrid zone, with low among-male variation consistent with strong post-copulatory sexual selection on sperm cells. We hypothesize that divergence in sperm length may constitute a reproductive barrier between species, as sperm length co-evolves with the size of specialized female sperm storage tubules. Sperm does not appear to act as a post-zygotic barrier, as sperm from hybrids was unexceptional.

Males were captured and measured for 13 plumage characters reflecting species identity; these were summed to give plumage index (see, e.g., Shriver, W.G., Gibbs, J.P., Vickery, P.D., Gibbs, H.L., Hodgman, T.P., Jones, P.T., Jacques, C.N., 2005. Concordance between morphological and molecular markers in assessing hybridization between sharp-tailed sparrows in New England. Auk 122, 94–107. https://doi.org/10.1642/0004-8038(2005)122[0094:CBMAMM]2.0.CO;2). Hybrid index was determined from 135 SNPs showing fixed differences between the species. The index reflects the proportion of these SNPs with the saltmarsh sparrow genotype. Genotyping was performed with ddRAD sequencing. Sperm morphology was measured for three sperm segments which sum to total sperm length, using light microscopy. Up to 10 morphologically normal cells were measured per male, depending on cell availability.