Spermatozoa are among the most diversified cells in the animal kingdom, but the underlying evolutionary forces affecting intraspecific variation in sperm morphology are poorly understood. It has been hypothesized that sperm competition is a potent selection pressure on sperm variation within species. Here, we examine intraspecific variation in total sperm length of 22 wild passerine bird species (21 genera, 11 families) in relation to the risk of sperm competition, as expressed by the frequency of extrapair paternity and relative testis size. We demonstrate, by using phylogenetic comparative methods, that between-male variation in sperm length within species is closely and negatively linked to the risk of sperm competition. This relationship was even stronger when only considering species in which data on sperm length and extrapair paternity originated from the same populations. Intramale variation in sperm length within species was also negatively, although nonsignificantly, related to sperm competition risk. Our findings suggest that postcopulatory sexual selection is a powerful evolutionary force reducing the intraspecific phenotypic variation in sperm-size traits, potentially driving the diversification of sperm morphology across populations and species.

Sperm swimming speed is an important determinant of male fertility and sperm competitiveness. Despite its fundamental biological importance, the underlying evolutionary processes affecting this male reproductive trait are poorly understood. Using a comparative approach in a phylogenetic framework, we tested the predictions that sperm swim faster with (1) increased risk of sperm competition, (2) shorter duration of female sperm storage, and (3)

BackgroundThe rate of extrapair paternity is a commonly used index for the risk of sperm competition in birds, but paternity data exist for only a few percent of the approximately 10400 extant species. As paternity analyses require extensive field sampling and costly lab work, species coverage in this field will probably not improve much in the foreseeable future. Recent findings from passerine birds, which constitute the largest avian order (∼5 900 species), suggest that sperm phenotypes carry a signature of sperm competition. Here we examine how well standardized measures of sperm length variation can predict the rate of extrapair paternity in passerine birds.Methodology/Principal FindingsWe collected sperm samples from 55 passerine species in Canada and Europe for which extrapair paternity rates were already available from either the same (n = 24) or a different (n = 31) study population. We measured the total length of individual spermatozoa and found that both the coefficient of between-male variation (CVbm) and within-male variation (CVwm) in sperm length were strong predictors of the rate of extrapair paternity, explaining as much as 65% and 58%, respectively, of the variation in extrapair paternity among species. However, only the CVbm predictor was independent of phylogeny, which implies that it can readily be converted into a currency of extrapair paternity without the need for phylogenetic correction.Conclusion/SignificanceWe propose the CVbm index as an alternative measure to extrapair paternity for passerine birds. Given the ease of sperm extraction from male birds in breeding condition, and a modest number of sampled males required for a robust estimate, this new index holds a great potential for mapping the risk of sperm competition across a wide range of passerine birds.

Rapid diversification of sexual traits is frequently attributed to sexual selection, though explicit tests of this hypothesis remain limited. Spermatozoa exhibit remarkable variability in size and shape, and studies report a correlation between sperm morphology (sperm length and shape) and sperm competition risk or female reproductive tract morphology. However, whether postcopulatory processes (e.g., sperm competition and cryptic female choice) influence the speed of evolutionary diversification in sperm form is unknown. Using passerine birds, we quantified evolutionary rates of sperm length divergence among lineages (i.e., species pairs) and determined whether these rates varied with the level of sperm competition (estimated as relative testes mass). We found that relative testes mass was significantly and positively associated with more rapid phenotypic divergence in sperm midpiece and flagellum lengths, as well as total sperm length. In contrast, there was no association between relative testes mass and rates of evolutionary divergence in sperm head size, and models suggested that head length is evolutionarily constrained. Our results are the first to show an association between the strength of sperm competition and the speed of sperm evolution, and suggest that postcopulatory sexual selection promotes rapid evolutionary diversification of sperm morphology.

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Mitochondrial DNA usually shows low sequence variation within and high sequence divergence among species, which makes it a useful marker for phylogenetic inference and DNA barcoding. A previous study on the common redstart (Phoenicurus phoenicurus) revealed two very different mtDNA haplogroups (5% K2P distance). This divergence is comparable to that among many sister species; however, both haplogroups coexist and interbreed in Europe today. Herein, we describe the phylogeographic pattern of these lineages and test hypotheses for how such high diversity in mtDNA has evolved. We found no evidence for mitochondrial pseudogenes confirming that both haplotypes are of mitochondrial origin. When testing for possible reproductive barriers, we found no evidence for lineage-specific assortative mating and no difference in sperm morphology, indicating that they are not examples of cryptic species, nor likely to reflect the early stages of speciation. A gene tree based on a short fragment of cytochrome c oxidase subunit 1 from the common redstart and 10 other Phoenicurus species, showed no introgression from any of the extant congenerics. However, introgression from an extinct congeneric cannot be excluded. Sequences from two nuclear introns did not show a similar differentiation into two distinct groups. Mismatch distributions indicated that the lineages have undergone similar demographic changes. Taken together, these results confirm that deeply divergent mitochondrial lineages can coexist in biological species. Sympatric mtDNA divergences are relatively rare in birds, but the fact that they occur argues against the use of threshold mtDNA divergences in species delineation.

Spermatozoa exhibit considerable interspecific variability in size and shape. Our understanding of the adaptive significance of this diversity, however, remains limited. Determining how variation in sperm structure translates into variation in sperm performance will contribute to our understanding of the evolutionary diversification of sperm form. Here, using data from passerine birds, we test the hypothesis that longer sperm swim faster because they have more available energy. We found that sperm with longer midpieces have higher levels of intracellular adenosine triphosphate (ATP), but that greater energy reserves do not translate into faster-swimming sperm. Additionally, we found that interspecific variation in sperm ATP concentration is not associated with the level of sperm competition faced by males. Finally, using Bayesian methods, we compared the evolutionary trajectories of sperm morphology and ATP content, and show that both traits have undergone directional evolutionary change. However, in contrast to recent suggestions in other taxa, we show that changes in ATP are unlikely to have preceded changes in morphology in passerine sperm. These results suggest that variable selective pressures are likely to have driven the evolution of sperm traits in different taxa, and highlight fundamental biological differences between taxa with internal and external fertilization, as well as those with and without sperm storage.

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