Sperm performance in conspecific and heterospecific female fluid

Abstract

Divergent sexual selection within allopatric populations may result in divergent sexual phenotypes, which can act as reproductive barriers between populations upon secondary contact. This hypothesis has been most tested on traits involved in precopulatory sexual selection, with less work focusing on traits that act after copulation and before fertilization (i.e., postcopulatory prezygotic traits), particularly in internally fertilizing vertebrates. However, postcopulatory sexual selection within species can also drive trait divergence, resulting in reduced performance of heterospecific sperm within the female reproductive tract. Such incompatibilities, arising as a by-product of divergent postcopulatory sexual selection in allopatry, can represent reproductive barriers, analogous to species-assortative mating preferences. Here, we tested for postcopulatory prezygotic reproductive barriers between three pairs of taxa with diverged sperm phenotypes and moderate-to-high opportunity for postcopulatory sexual selection (barn swallows Hirundo rustica versus sand martins Riparia riparia, two subspecies of bluethroats, Luscinia svecica svecica versus L. s. namnetum, and great tits Parus major versus blue tits Cyanistes caeruleus). We tested sperm swimming performance in fluid from the outer reproductive tract of females, because the greatest reduction in sperm number in birds occurs as sperm swim across the vagina. Contrary to our expectations, sperm swam equally well in fluid from conspecific and heterospecific females, suggesting that postcopulatory prezygotic barriers do not act between these taxon pairs, at this stage between copulation and fertilization. We therefore suggest that divergence in sperm phenotypes in allopatry is insufficient to cause widespread postcopulatory prezygotic barriers in the form of impaired sperm swimming performance in passerine birds.

Keywords: Cryptic female choice; postcopulatory prezygotic barriers; sexual selection; speciation; sperm competition; sperm motility; sperm velocity.

Figure 1

Summary of methods. (A) The female reproductive tract was washed with a phosphate‐buffered saline (PBS) solution. These samples were frozen for later use. (B) Ejaculates were collected via cloacal massage. (C) Sperm and thawed (prewarmed) female fluids were mixed, as follows. The ejaculate was diluted in PBS to create a stock solution; 2.5 μL of this stock solution was added to each of three experimental treatments (control PBS; a conspecific female fluid, and a heterospecific female fluid). Treatments were loaded onto a four‐chambered microscope slide and filmed (small gray boxes and solid arrows, showing eight filming locations). As per Appendix 1, the number of filming locations varied among species pairs. The stock solution was not filmed in tit experiments. The assignment of female species to chamber was rotated among blocks. Sp = species

Figure 2

Change in sperm swimming parameters (A, B: sperm velocity, VCL, μm/S; C, D: proportion of motile sperm) over time in barn swallows (A, C) and sand martins (B, D). Sperm were mixed with fluid from the reproductive tract of barn swallow females (tan), sand martin females (blue) or a control saline solution (gray) before being filmed. (A, B) Sand martin sperm swimming speed increased over time more than did barn swallow sperm swimming speed, and barn swallow female fluid supported a faster increase in sperm swimming speed for males of both species. (C, D) The proportion of motile cells decreased over time, and was lower in the control treatment than in female fluids, for both male species. Decrease over time was faster in sand martin ejaculates than in barn swallow ejaculates. Plots drawn using ggplot2 using raw data; shading indicates 95% confidence intervals; statistical tests were performed with both male species considered simultaneously, although they are drawn separately here.

Figure 3

Effect of experimental treatments on sperm swimming parameters for two subspecies of bluethroats (A: L. s. namnetum; B: L. s. svecica; C: both subspecies). A, B: sperm velocity was measured over time fluid samples from the reproductive tract of namnetum females (tan) and svecica females (blue), or control (gray). Sperm velocity decreased over time, but this decrease was not related to either male or female subspecies. (C) The proportion of motile cells was measured in conspecific and heterospecific female fluids and in control saline solution (PBS), and could not be analyzed with respect to time because of the distribution of the data. Plots A and B drawn using ggplot2 using raw data; shading indicates 95% confidence intervals; statistical tests were performed with both male species considered simultaneously, although they are drawn separately here.

Figure 4

Change in sperm swimming parameters (A, B: sperm velocity, VCL, μm/S; C, D: proportion of motile sperm) over time in great tits (A, C) and blue tits (B, D). Sperm were mixed with fluid from the reproductive tract of blue tit females (blue), great tit females (tan) or a control saline solution (gray) before being filmed. (A, B) For sperm of both species, swimming speed decreased more quickly in the control treatment than in either female fluid. The initial swimming speed of sperm was lower in great tit female fluid than in other treatments. (C, D) Proportion of motile sperm decreased over time, with the decrease being faster in blue tit males than great tit males. Plots drawn using ggplot2 using raw data; shading indicates 95% confidence intervals; statistical tests were performed with both male species considered simultaneously, although they are drawn separately here.