Sperm head morphology is associated with sperm swimming speed: A comparative study of songbirds using electron microscopy

Sperm exhibit extraordinary levels of morphological diversification across the animal kingdom. In songbirds, sperm have a helically shaped head incorporating a distinct acrosomal membrane or “helical keel,” the form and extent of which varies across species. The functional significance of this helical shape, however, remains unknown. Using scanning electron microscopy, we quantified inter‐ and intraspecific variation in sperm head morphology across 36 songbird species (Passeriformes: Passerida). Using phylogenetic comparative methods, we investigated the relationship between sperm head morphology and both sperm swimming speed and the frequency of extra‐pair young (EPY). We found that species whose sperm had a relatively more pronounced helical form (i.e., long acrosome, short nucleus, wide helical membrane, and a more pronounced waveform along the sperm head “core”) had faster‐swimming sperm. We found no evidence of a relationship between interspecific variation in sperm head morphology and EPY, although we did find that among‐ and within‐male variation in sperm head traits were negatively correlated with EPY. Applying principles of fluid mechanics, we discuss how the helical form of the sperm head may influence swimming speed, and suggest that further studies considering aspects of sperm morphology beyond sperm length are needed to improve our understanding of sperm structure‐function relationships.     

Postcopulatory sexual selection is associated with accelerated evolution of sperm morphology

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.     

Patterns of sperm damage in Chernobyl passerine birds suggest a trade-off between sperm length and integrity

Interspecific variation in sperm size is enigmatic, but generally assumed to reflect species-specific trade-offs in selection pressures. Among passerine birds, sperm length varies sevenfold, and sperm competition risk seems to drive the evolution of longer sperm. However, little is known about factors favouring short sperm or constraining the evolution of longer sperm. Here, we report a comparative analysis of sperm head abnormalities among 11 species of passerine bird in Chernobyl, presumably resulting from chronic irradiation following the 1986 accident. Frequencies of sperm abnormalities varied between 15.7 and 77.3% among species, more than fourfold higher than in uncontaminated areas. Nonetheless, species ranked similarly in sperm abnormalities in unpolluted areas as in Chernobyl, pointing to intrinsic factors underlying variation in sperm damage among species. Scanning electron microscopy of abnormal spermatozoa revealed patterns of acrosome damage consistent with premature acrosome reaction. Sperm length, but not sperm competition risk explained variation in sperm damage among species. This suggests that longer spermatozoa are more susceptible to premature acrosome reaction. Therefore, we hypothesize a trade-off between sperm length and sperm integrity affecting sperm evolution in passerine birds.     

Sperm midpiece length predicts sperm swimming velocity in house mice

Evolutionary biologists have argued that there should be a positive relationship between sperm size and sperm velocity, and that these traits influence a male''s sperm competitiveness. However, comparative analyses investigating the evolutionary associations between sperm competition risk and sperm morphology have reported inconsistent patterns of association, and in vitro sperm competition experiments have further confused the issue; in some species, males with longer sperm achieve more competitive fertilization, while in other species males with shorter sperm have greater sperm competitiveness. Few investigations have attempted to address this problem. Here, we investigated the relationship between sperm morphology and sperm velocity in house mice (Mus domesticus). We conducted in vitro sperm velocity assays on males from established selection lines, and found that sperm midpiece size was the only phenotypic predictor of sperm swimming velocity.     

Intraspecific variation in sperm length is negatively related to sperm competition in passerine birds

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 morphology and evidence for sperm competition among parrots

Sperm competition is an important component of post‐copulatory sexual selection that has shaped the evolution of sperm morphology. Previous studies have reported that sperm competition has a concurrently directional and stabilizing effect on sperm size. For example, bird species that show higher levels of extrapair paternity and larger testes (proxies for the intensity of sperm competition) have longer sperm and lower coefficients of variation in sperm length, both within and between males. For this reason, these sperm traits have been proposed as indexes to estimate the level of sperm competition in species for which other measures are not available. The relationship between sperm competition and sperm morphology has been explored mostly for bird species that breed in temperate zones, with the main focus on passerine birds. We measured sperm morphology in 62 parrot species that breed mainly in the tropics and related variation in sperm length to life‐history traits potentially indicative of the level of sperm competition. We showed that sperm length negatively correlated with the within‐male coefficient of variation in sperm length and positively with testes mass. We also showed that sperm is longer in sexually dichromatic and in gregarious species. Our results support the general validity of the hypothesis that sperm competition drives variation in sperm morphology. Our analyses suggest that post‐copulatory sexual selection is also important in tropical species, with more intense sperm competition among sexually dichromatic species and among species that breed at higher densities.     

The evolution of sperm morphometry in pheasants

Post-copulatory sexual selection is thought to be a potent evolutionary force driving the diversification of sperm shape and function across species. In birds, insemination and fertilization are separated in time and sperm storage increases the duration of sperm-female interaction and hence the opportunity for sperm competition and cryptic female choice. We performed a comparative study of 24 pheasant species (Phasianidae, Galliformes) to establish the relative importance of sperm competition and the duration of sperm storage for the evolution of sperm morphometry (i.e. size of different sperm traits). We found that sperm size traits were negatively associated with the duration of sperm storage but were independent of the risk of sperm competition estimated from relative testis mass. Our study emphasizes the importance of female reproductive biology for the evolution of sperm morphometry particularly in sperm-storing taxa.     

Evolution of sperm structure and energetics in passerine birds

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.     

Early diversification of sperm size in the evolutionary history of the old world leaf warblers (Phylloscopidae)

Sperm morphological traits are highly variable among species and are commonly thought to evolve by post‐copulatory sexual selection. However, little is known about the evolutionary dynamics of sperm morphology, and whether rates of evolutionary change are variable over time and among taxonomic groups. Here, we examine sperm morphology from 21 species of Old World leaf warblers (Phylloscopidae), a group of generally dull, sexually monochromatic birds, which are known to have high levels of extra‐pair paternity. We found that sperm length differs markedly across species, spanning about 40% of the range observed across a larger selection of passerine birds. Furthermore, we found strong support for an ‘early‐burst’ model of trait evolution, implying that the majority of divergence in sperm length has occurred early in the evolutionary history of this clade with subsequent evolutionary stasis. This large early divergence matches the early divergence reported in ecological traits (i.e. body size and feeding behaviour). Our findings demonstrate that rates of evolution in sperm morphology can change over time in passerine taxa, and that evolutionary stasis in sperm traits can occur even in species exhibiting characteristics consistent with moderate‐to‐high levels of sperm competition. It remains a major challenge to identify the selection mechanisms and possible constraints responsible for these variable rates of sperm evolution.     

Sperm competition and the evolution of the sperm hook in house mice

Sperm morphology varies considerably both between and within species. The sperm of many muroid rodents bear an apical hook at the proximal end of the head. The curvature of the sperm hook varies greatly across species, however the adaptive significance of the sperm hook is currently not known. In wood mice the apical hooks intertwine to form sperm ‘trains’, which exhibit faster swimming velocities than single cells. Thus, it has been suggested that if sperm ‘trains’ were advantageous in a competitive situation, then the apical sperm hook might be an evolutionary product of selection via sperm competition. A comparative study of rodent species provided support for the hypothesis, and showed that species with higher levels of sperm competition had more reflected sperm hooks. Here, we tested this hypothesis at the intraspecific level. We quantified sperm hook morphology from seven house mouse populations, and found that interpopulation variation in hook curvature was not explained by variation in sperm competition risk. Furthermore, observations of ejaculated sperm revealed that sperm groups are not a common characteristic of mouse ejaculates. We suggest that selection for sperm attachment to the oviduct epithelium, and thus better retainment of sperm fertilizing potential, may provide a more general explanation of the evolutionary relationship between sperm competition risk and the curvature of the sperm hook among rodents, and provide a phylogenetic comparison among rodent species that supports our hypothesis.     

Sexual selection and the evolution of sperm morphology in sharks

Post‐copulatory sexual selection, and sperm competition in particular, is a powerful selective force shaping the evolution of sperm morphology. Although mounting evidence suggests that post‐copulatory sexual selection influences the evolution of sperm morphology among species, recent evidence also suggests that sperm competition influences variation in sperm morphology at the intraspecific level. However, contradictory empirical results and limited taxonomic scope have led to difficulty in assessing the generality of sperm morphological responses to variation in the strength of sperm competition. Here, we use phylogenetically controlled analyses to explore the effects of sperm competition on sperm morphology and variance in sharks, a basal vertebrate group characterized by wide variation in rates of multiple mating by females, and consequently sperm competition risk. Our analyses reveal that shark species experiencing greater levels of sperm competition produce sperm with longer flagella and that sperm flagellum length is less variable in species under higher sperm competition risk. In contrast, neither the length of the sperm head and midpiece nor variation in sperm head and midpiece length was associated with sperm competition risk. Our findings demonstrate that selection influences both the inter‐ and intraspecific variation in sperm morphology and suggest that the flagellum is an important target of sexual selection in sharks. These findings provide important insight into patterns of selection on the ejaculate in a basal vertebrate lineage.     

Polyandry reduces sperm length variation in social insects

Postcopulatory sexual selection, either in the form of sperm competition or cryptic female choice, is an important selective force that is thought to have generated the enormous variation in sperm morphology observed interspecifically. However, the evolutionary significance of intraspecific variation in sperm morphology, and the role that postcopulatory sexual selection plays in influencing this variation, remains poorly investigated in invertebrates. Here, we tested the hypothesis that postcopulatory sexual selection reduces variation in sperm morphology, both between and within males, in 27 species of eusocial ants and bees. These eusocial species offer an unusual opportunity to assess how selection acts on variance in sperm morphology, as haploid males produce clonal, haploid sperm that does not experience haploid-diploid conflict. We provide solid evidence that males of polyandrous ant and bee species indeed produce less-variable sperm, indicating that sperm competition selected for sperm of superior quality. Our results offer a mechanistic explanation for the evolution of high-quality sperm and provide comprehensive evidence that sperm morphology of social insects is influenced by sexual selection.     

Convergence, recurrence and diversification of complex sperm traits in diving beetles (Dytiscidae)

Sperm display remarkable morphological diversity among even closely related species, a pattern that is widely attributed to postcopulatory sexual selection. Surprisingly few studies have used phylogenetic analyses to discern the details of evolutionary diversification in ornaments and armaments subject to sexual selection, and the origins of novel sperm traits and their subsequent modification are particularly poorly understood. Here we investigate sperm evolution in diving beetles (Dytiscidae), revealing dramatic diversification in flagellum length, head shape, presence of sperm heteromorphism, and the presence/type of sperm conjugation, an unusual trait where two or more sperm unite for motility or transport. Sperm conjugation was found to be the ancestral condition in diving beetles, with subsequent diversification into three forms, each exhibiting varying degrees of evolutionary loss, convergence, and recurrence. Sperm head shape, but not length or heteromorphism, was found to evolve in a significantly correlated manner with conjugation, consistent with the different mechanisms of head alignment and binding required for the different forms of conjugation. Our study reveals that sperm morphological evolution is channeled along particular evolutionary pathways (i.e., conjugate form), yet subject to considerable diversification within those pathways through modification in sperm length, head shape, and heteromorphism.     

Sperm morphology and sperm velocity in passerine birds

Sperm velocity is one of the main determinants of the outcome of sperm competition. Since sperm vary considerably in their morphology between and within species, it seems likely that sperm morphology is associated with sperm velocity. Theory predicts that sperm velocity may be increased by enlarged midpiece (energetic component) or flagellum length (kinetic component), or by particular ratios between sperm components, such as between flagellum length and head size. However, such associations have rarely been found in empirical studies. In a comparative framework in passerine birds, we tested these theoretical predictions both across a wide range of species and within a single family, the New World blackbirds (Icteridae). In both study groups, sperm velocity was influenced by sperm morphology in the predicted direction. Consistent with theoretical models, these results show that selection on sperm morphology and velocity are likely to be concomitant evolutionary forces.     

Long sperm fertilize more eggs in a bird

Sperm competition, in which the ejaculates of multiple males compete to fertilize a female''s ova, results in strong selection on sperm traits. Although sperm size and swimming velocity are known to independently affect fertilization success in certain species, exploring the relationship between sperm length, swimming velocity and fertilization success still remains a challenge. Here, we use the zebra finch (Taeniopygia guttata), where sperm size influences sperm swimming velocity, to determine the effect of sperm total length on fertilization success. Sperm competition experiments, in which pairs of males whose sperm differed only in length and swimming speed, revealed that males producing long sperm were more successful in terms of (i) the number of sperm reaching the ova and (ii) fertilizing those ova. Our results reveal that although sperm length is the main factor determining the outcome of sperm competition, complex interactions between male and female reproductive traits may also be important. The mechanisms underlying these interactions are poorly understood, but we suggest that differences in sperm storage and utilization by females may contribute to the outcome of sperm competition.     

Distinct evolutionary patterns of morphometric sperm traits in passerine birds

The striking diversity of sperm shape across the animal kingdom is still poorly understood. Postcopulatory sexual selection is an important factor driving the evolution of sperm size and shape. Interestingly, morphometric sperm traits, such as the length of the head, midpiece and flagellum, exhibit a strong positive phenotypic correlation across species. Here we used recently developed comparative methods to investigate how such phenotypic correlations between morphometric sperm traits may evolve. We compare allometric relationships and evolutionary trajectories of three morphometric sperm traits (length of head, midpiece and flagellum) in passerine birds. We show that these traits exhibit strong phenotypic correlations but that allometry varies across families. In addition, the evolutionary trajectories of the midpiece and flagellum are similar while the trajectory for head length differs. We discuss our findings in the light of three scenarios accounting for correlated trait evolution: (i) genetic correlation; (ii) concerted response to selection acting simultaneously on different traits; and (iii) phenotypic correlation between traits driven by mechanistic constraints owing to selection on sperm performance. Our results suggest that concerted response to selection is the most likely explanation for the phenotypic correlation between morphometric sperm traits.     

Rapid evolution of testis size relative to sperm morphology suggests that post‐copulatory selection targets sperm number in Anolis lizards

Post‐copulatory sexual selection is thought to be responsible for much of the extraordinary diversity in sperm morphology across metazoans. However, the extent to which post‐copulatory selection targets sperm morphology versus sperm production is generally unknown. To address this issue, we simultaneously characterized the evolution of sperm morphology (length of the sperm head, midpiece and flagellum) and testis size (a proxy for sperm production) across 26 species of Anolis lizards, a group in which sperm competition is likely. We found that the length of the sperm midpiece has evolved 2–3 times faster than that of the sperm head or flagellum, suggesting that midpiece size may be the most important aspect of sperm morphology with respect to post‐copulatory sexual selection. However, testis size has evolved faster than any aspect of sperm morphology or body size, supporting the hypothesis that post‐copulatory sexual selection acts more strongly upon sperm production than upon sperm morphology. Likewise, evolutionary increases in testis size, which typically indicate increased sperm competition, are not associated with predictable changes in sperm morphology, suggesting that any effects of post‐copulatory selection on sperm morphology are either weak or variable in direction across anoles. Collectively, our results suggest that sperm production is the primary target of post‐copulatory sexual selection in this lineage.     

Novelty and emergent patterns in sperm: Morphological diversity and evolution of spermatozoa and sperm conjugation in ground beetles (Coleoptera: Carabidae)

The beetle family Carabidae, with about 40,000 species, exhibits enough diversity in sperm structure and behavior to be an excellent model system for studying patterns and processes of evolution. We explore their potential, documenting sperm form in 177 species of ground beetles using light microscopy and collecting data on one qualitative and seven quantitative phenotypic traits. Our sampling captures 61% of the tribal-level diversity of ground beetles. These data highlight the notable morphological diversity of sperm in ground beetles and suggest that sperm in the group have dynamic evolutionary histories with much morphological innovation and convergence. Sperm vary among species in total length (48–3,400 μm), head length (0.5–270 μm), and head width (0.2–6.3 μm). Most ground beetles make sperm with heads that are indistinct from the flagella at the gross morphological level. However, some or all Omophron, Trachypachus, and Dyschiriini make broad-headed sperm that show morphological differences between species. Most ground beetles package their sperm into groups of sperm, termed conjugates, and ground beetles show variation in conjugate form and in the number and arrangement of sperm in a conjugate. Most ground beetles make sperm conjugates by embedding their sperm in a hyaline rod or spermatostyle. The spermatostyle is remarkably variable among species and varies in length from 17 to 41,000 μm. Several unrelated groups of ground beetles make only singleton sperm, including Nebriinae, Cicindelinae, many Trechinae, and the tribe Paussini. In order to study patterns in sperm evolution, we combine these data with a low-resolution phylogeny of ground beetles. Results from modern comparative analyses suggest the following: (a) sperm differ from conjugates in some aspect of their underlying evolutionary process, (b) sperm have influenced conjugate evolution and vice versa, and (c) conjugation with a spermatostyle likely evolved early within the history of Carabidae and it has been lost independently at least three times.     

No evidence of sperm conjugate formation in an Australian mouse bearing sperm with three hooks

Sperm conjugation occurs when two or more sperm physically unite for motility or transport through the female reproductive tract. In many muroid rodent species, sperm conjugates have been shown to form by a single, conspicuous apical hook located on the sperm head. These sperm “trains” have been reported to be highly variable in size and, despite all the heads pointing in roughly the same direction, exhibit a relatively disordered arrangement. In some species, sperm “trains” have been shown to enhance sperm swimming speed, and thus have been suggested to be advantageous in sperm competition. Here, we assessed the behavior of sperm in the sandy inland mouse (Pseudomys hermannsburgensis), a muroid rodent that bears sperm with three apical hooks. First, we accrued genetic evidence of multiple paternity within “wild” litters to unequivocally show that sperm competition does occur in this species. Following this we utilized both in vitro and in vivo methodologies to determine whether sandy inland mouse sperm conjugate to form motile trains. Our observations of in vitro preparations of active sperm revealed that sandy inland mouse sperm exhibit rapid, progressive motility as individual cells only. Similarly, histological sections of the reproductive tracts of mated females revealed no in vivo evidence of sperm conjugate formation. We conclude that the unique, three‐hooked morphology of the sandy inland mouse sperm does not facilitate the formation of motile conjugates, and discuss our findings in relation to the different hypotheses for the evolution of the muroid rodent hook/s.     

Intra‐specific variance in sperm morphometry: a comparison between wild and domesticated Zebra Finches Taeniopygia guttata

The Zebra Finch Taeniopygia guttata is a model bird species for the experimental study of behavioural and evolutionary concepts in captivity and especially sexual selection. The validity of sexual selection studies of domesticated birds is of long‐standing concern as little is known about the influence of domestication on sexually selected traits. Most domesticated Zebra Finch populations are maintained under a strict breeding regime to avoid potential inbreeding. However, these breeding regimes may interfere with the processes of sexual selection and influence the evolution of sexually selected traits because they may limit or prohibit active mate choice. Here, we investigated the potential impact of a monogamous breeding scheme in a domesticated population in which active mate choice is largely inhibited, on the evolution of sperm morphometry as a sexually selected trait. We compared sperm morphometric traits (total sperm length and length of sperm head, midpiece and flagellum), and the variance thereof, between a domesticated and two wild Zebra Finch populations. Although we found significant differences between the three populations for certain sperm traits (head length, midpiece length), which may be of importance in postcopulatory sexual selection, overall, variance in sperm morphometry did not differ between the domesticated and the wild Zebra Finch populations. Our results validate the use of domesticated Zebra Finches for further studies of postcopulatory sexual selection and sperm competition.