Molecular Diversification of the Seminal Fluid Proteome in a Recently Diverged Passerine Species Pair

Abstract

Seminal fluid proteins (SFPs) mediate an array of postmating reproductive processes that influence fertilization and fertility. As such, it is widely held that SFPs may contribute to postmating, prezygotic reproductive barriers between closely related taxa. We investigated seminal fluid (SF) diversification in a recently diverged passerine species pair (Passer domesticus and Passer hispaniolensis) using a combination of proteomic and comparative evolutionary genomic approaches. First, we characterized and compared the SF proteome of the two species, revealing consistencies with known aspects of SFP biology and function in other taxa, including the presence and diversification of proteins involved in immunity and sperm maturation. Second, using whole-genome resequencing data, we assessed patterns of genomic differentiation between house and Spanish sparrows. These analyses detected divergent selection on immunity-related SF genes and positive selective sweeps in regions containing a number of SF genes that also exhibited protein abundance diversification between species. Finally, we analyzed the molecular evolution of SFPs across 11 passerine species and found a significantly higher rate of positive selection in SFPs compared with the rest of the genome, as well as significant enrichments for functional pathways related to immunity in the set of positively selected SF genes. Our results suggest that selection on immunity pathways is an important determinant of passerine SF composition and evolution. Assessing the role of immunity genes in speciation in other recently diverged taxa should be prioritized given the potential role for immunity-related proteins in reproductive incompatibilities in Passer sparrows.

Keywords: cryptic female choice; fertility; immunity; positive selection; reproduction; selective sweep; sperm competition.

Fig. 1.

Distribution and sampling site of the house and Spanish sparrow. Blue indicates the distribution of the house sparrow and red indicates the distribution of the Spanish sparrow. Hatched areas indicate sympatric regions where the house and Spanish sparrow distribution overlap. The yellow color indicates the distribution of the hybrid homoploid species, the Italian sparrow (not investigated in this study). White dot and arrow indicate the sampling location for seminal fluid samples used in this study.

Fig. 2.

Comparison of the house sparrow and Spanish sparrow seminal fluid proteomes. (A) One-dimensional (1D) gels of house sparrow (left) and Spanish sparrow (right) seminal fluid samples. Gels were loaded with 9 μg/lane of protein and stained with silver stain. NB, gel banding patterns were consistent across biological replicates in both species (replicate gels not shown). (B) Correlation in the abundance of proteins identified in both the house sparrow and Spanish sparrow proteomes. Protein abundance is plotted as log 10 transformed APEX values, which represent normalized estimates of protein abundance for 467 shared proteins. Correlation coefficient is Spearman rank correlation.

Fig. 3.

MA plot comparing protein abundances between the house and Spanish sparrow seminal fluid proteomes. Differential abundance analysis for proteins identified in both species revealed 61 significant proteins after Benjamini–Hochberg multiple testing correction. Proteins of significantly greater abundance in the house sparrow are shown in blue, whereas proteins of significantly greater abundance in the Spanish sparrow are shown in red, and nonsignificant proteins are in gray. Proteins with P values <0.001 have been labeled with protein names.

Fig. 4.

Patterns of genomic differentiation and divergence and signatures of selection in SF proteome genes between house and Spanish sparrows. Red dashed lines represent midpoint of genes of interest on chromosomes 11, 1, 4, and 8, respectively. Differentiation and divergence was measured as F_ST and d_XY, and nucleotide diversity measured as π (blue for house sparrow and red for Spanish sparrow). Extended haplotype homozygosity (xpEHH) statistics show evidence of recent selective sweeps. Horizontal dashed line represents threshold of significance equivalent to P value of 0.001.

Fig. 5.

Protein–protein interaction network for genes showing evidence of positive selection. Genes exhibiting signature of positive selection (i.e., comparison of M8 and M8a site models) were analyzed using the STRING protein–protein interaction database, allowing for up to five additional protein interactors. Network nodes represent proteins and edges represent protein–protein associations. Different colored edges represent complementary types of empirical evidence supporting interactions, including evidence from curated databases, experimentally determined interactions, and coexpression studies. The protein network exhibited significantly greater connectivity that expected (PPI enrichment P < 1.0e-16). Proteins linked to immunity form a cluster (*) including several complement proteins (e.g., C5 and C2), whereas the cluster on the right (**) includes several proteins involved in sperm–egg interactions (e.g., TCP1 and CCT5).