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.     

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.     

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.     

Intra‐ and intermale variability of mature sperm traits analysed in two brackish water populations of the pipefish Syngnathus abaster (Syngnathidae)

Sperm cells are highly diversified in animals, and considerable research effort has focused on variation in sperm morphology among species. Surprisingly, little is known about intraspecific variation in sperm morphology. We analysed within‐ and between‐male variation in mature sperm traits in two brackish water populations of the pipefish Syngnathus abaster. Four morphometric parameters, such as the width and length of the head (including nucleus, and midpiece), length of flagellum and total sperm length were taken into account. The differences in all morphometric parameters analysed between populations were not statistically significant. Moreover, the multidimensional scaling analysis shows that (i) the two populations seem to be indistinguishable based on their spermatozoa and (ii) there is not polymorphism, being sperm not distinguishable into discrete classes both within a single male and between males of each populations. The latter datum does not seem to support the presence of polymorphic sperm in syngnathids. Both populations, however, exhibit a high variation in all sperm traits, both among individual sperm within an ejaculate and among males within each population. The relationship between sperm traits variability and the low selection pressure determined by the absence of postcopulatory sexual selection (i.e. absence of sperm competition) is discussed.     

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.     

Relationship between pre‐ and post‐copulatory traits in Salvator rufescens (Squamata: Teiidae)

Understanding pre‐ and post‐copulatory mechanisms of sexual selection can provide insights into the evolution of male reproductive strategies. The phenotype‐linked fertility hypothesis postulates that male sperm quality and secondary sexual characteristics will positively co‐vary, whereas the sperm competition hypothesis predicts a negative association between those traits. Male reproductive traits often show variation throughout the reproductive period, suggesting that the relationship between pre‐ and post‐copulatory sexual selection may vary temporally. Here, we evaluated the relationship between secondary sexual character and sperm traits and its temporal variation in Salvator rufescens, a south American lizard. We observed a negative relationship between jaw muscle and principal piece length of sperm and a variation in the relationship between pre‐ and post‐copulatory traits throughout the reproductive period. Collectively, our results evidenced a trade‐off between pre‐ and post‐copulatory traits and a strong seasonal flexibility of male reproductive strategies in this lizard species.     

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.     

Increased postcopulatory sexual selection reduces the intramale variation in sperm design

Sperm competition is an important force driving the evolution of sperm design and function. Inter- and intraspecific variation in sperm design are strongly influenced by the risk of sperm competition in many taxa. In contrast, the variation among sperm of one male (intramale variation) is less well understood. We investigated intramale variation in sperm design in passerine birds and found that risk of sperm competition is negatively associated with intramale variation. This result is the first clear evidence that variation among sperm within an individual male is influenced by postcopulatory sexual selection. Our finding has important implications for male traits under pre- and postcopulatory sexual selection.     

Postcopulatory Sexual Selection Results in Spermatozoa with More Uniform Head and Flagellum Sizes in Rodents

Interspecific comparative studies have shown that, in most taxa, postcopulatory sexual selection (PCSS) in the form of sperm competition drives the evolution of longer and faster swimming sperm. Work on passserine birds has revealed that PCSS also reduces variation in sperm size between males at the intraspecific level. However, the influence of PCSS upon intra-male sperm size diversity is poorly understood, since the few studies carried out to date in birds have yielded contradictory results. In mammals, PCSS increases sperm size but there is little information on the effects of this selective force on variations in sperm size and shape. Here, we test whether sperm competition associates with a reduction in the degree of variation of sperm dimensions in rodents. We found that as sperm competition levels increase males produce sperm that are more similar in both the size of the head and the size of the flagellum. On the other hand, whereas with increasing levels of sperm competition there is less variation in head length in relation to head width (ratio CV head length/CV head width), there is no relation between variation in head and flagellum sizes (ratio CV head length/CV flagellum length). Thus, it appears that, in addition to a selection for longer sperm, sperm competition may select more uniform sperm heads and flagella, which together may enhance swimming velocity. Overall, sperm competition seems to drive sperm components towards an optimum design that may affect sperm performance which, in turn, will be crucial for successful fertilization.     

Variation in sperm morphology among Afrotropical sunbirds

Birds show considerable variation in sperm morphology. Closely related species and subspecies can show diagnostic differences in sperm size. There is also variation in sperm size among males within a population, and recent evidence from passerine birds suggests that the coefficient of inter‐male variation in sperm length is negatively associated with the level of sperm competition. Here we examined patterns of inter‐ and intra‐specific variation in sperm length in 12 species of sunbird (Nectariniidae) from Nigeria and Cameroon, a group for which such information is extremely limited. We found significant variation among species in sperm total length, with mean values ranging from 74 μm to 116 μm, placing these species within the short to medium sperm length range for passerine birds. Most of this variation was explained by the length of the midpiece, which contains the fused mitochondria and is an important structure for sperm energetics. Relative midpiece length was negatively correlated with the coefficient of inter‐male variation in sperm total length across species, suggesting that sperm competition may have selected for greater midpiece length in this group. We also mapped sperm lengths onto a time‐calibrated phylogeny and found support for a phylogenetic signal in all sperm length components, except head length. A test of various evolutionary or tree transformation models gave strongest support for the Brownian motion model for all sperm components, i.e. divergences were best predicted by the phylogenetic distance between lineages. The coefficients of inter‐male variation in sperm total length indicate that sperm competition is high but variable among sunbird species, as is the case with passerine birds at large.     

No association between sperm competition and sperm length variation across dung flies (Scathophagidae)

Sperm length is extremely variable across species, but a general explanation for this variation is lacking. However, when the risk of sperm competition is high, sperm length is predicted to be less variable within species, and there is some evidence for this in birds and social insects. Here, we examined intraspecific variation in sperm length, both within and between males, and its potential associations with sperm competition risk and variation in female reproductive tract morphology across dung flies. We used two measures of variation in sperm size, and testis size was employed as our index of sperm competition risk. We found no evidence of associations between sperm length variation and sperm competition or female reproductive tract variation. These results suggest that variation in sperm competition risk may not always be associated with variation in sperm morphology, and the cause(s) of sperm length variation in dung flies remains unclear.     

No evidence for pre-copulatory sexual selection on sperm length in a passerine bird

There is growing evidence that post-copulatory sexual selection, mediated by sperm competition, influences the evolution of sperm phenotypes. Evidence for pre-copulatory sexual selection effects on sperm traits, on the other hand, is rather scarce. A recent paper on the pied flycatcher, Ficedula hypoleuca, reported phenotypic associations between sperm length and two sexually selected male traits, i.e. plumage colour and arrival date, thus invoking pre-copulatory sexual selection for longer sperm. We were unable to replicate these associations with a larger data set from the same and two additional study populations; sperm length was not significantly related to either male plumage colour or arrival date. Furthermore, there was no significant difference in sperm length between populations despite marked differences in male plumage colour. We also found some evidence against the previously held assumption of longer sperm being qualitatively superior; longer sperm swam at the same speed as shorter sperm, but were less able to maintain speed over time. We argue that both empirical evidence and theoretical considerations suggest that the evolution of sperm morphology is not primarily associated with pre-copulatory sexual selection on male secondary sexual traits in this or other passerine bird species. The relatively large between-male variation in sperm length in this species is probably due to relaxed post-copulatory sexual selection.     

Mammalian sperm and oviducts are sexually selected: evidence for co-evolution

Oviduct length was measured in 48 species representing 33 genera of mammals in order to examine possible relationships between female morphology and the occurrence of sperm competition due to matings with multiple males. Multiple regression analyses revealed that residuals of oviduct length were positively correlated both with residuals of testes weight and with sperm midpiece volume in the genera and species studied. These correlations remained significant after application of comparative analysis of independent contrasts to control for possible phylogenetic biases in the data set. These results indicate that sexual selection (relating to sperm competition and cryptic female choice) has influenced co-evolution of oviduct length, testes size and sperm morphology in mammals.     

Maintenance of sperm variation in a highly promiscuous wild bird

Postcopulatory sexual selection is an important force in the evolution of reproductive traits, including sperm morphology. In birds, sperm morphology is known to be highly heritable and largely condition-independent. Theory predicts, and recent comparative work corroborates, that strong selection in such traits reduces intraspecific phenotypic variation. Here we show that some variation can be maintained despite extreme promiscuity, as a result of opposing, copulation-role-specific selection forces. After controlling for known correlates of siring success in the superb fairy-wren (Malurus cyaneus), we found that (a) lifetime extra-pair paternity success was associated with sperm with a shorter flagellum and relatively large head, and (b) males whose sperm had a longer flagellum and a relatively smaller head achieved higher within-pair paternity. In this species extrapair copulations occur in the same morning, but preceding, pair copulations during a female's fertile period, suggesting that shorter and relatively larger-headed sperm are most successful in securing storage (defense), whereas the opposite phenotype might be better at outcompeting stored sperm (offense). Furthermore, since cuckolding ability is a major contributor to differential male reproductive output, stronger selection on defense sperm competition traits might explain the short sperm of malurids relative to other promiscuous passerines.     

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.     

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.     

Selection on sperm morphology under relaxed sperm competition in a wild passerine bird

Theories regarding the role of sexual selection on the evolution of sperm traits are based on an association between pre-copulatory (e.g. female preference) and post-copulatory (e.g. ejaculate quality) male reproductive traits. In tests of these hypotheses, sperm morphology has rarely been used, despite its high heritability and intra-individual consistency. We found evidence of selection for longer sperm through positive phenotypic associations between sperm size and the two major female preference traits in the pied flycatcher, Ficedula hypoleuca. Our results support the sexually selected sperm hypothesis in a species under low sperm competition and demonstrate that natural and pre-copulatory sexual selection forces should not be overlooked in studies of intraspecific sperm morphology evolution.     

Intra-individual variation in sperm tail length in murine rodents

In eutherian mammals, there are marked interspecific differences in sperm head shape and tail length. In a few species, sperm head variability occurs but intra-individual variation in sperm tail length has rarely been investigated or commented upon. Here, we ask the question: Do murine rodent species that have variable sperm head shapes exhibit greater intra-individual variation in sperm midpiece and total tail lengths than closely related species where little, or no, sperm head variability occurs? From three separate lineages, we selected three pairs of murine rodents, one of which has monomorphic, and the other variable, sperm head shape. These were from southern Asia the bandicoot rats Bandicota bengalensis and Bandicota indica , from southern Africa the veld rats, Aethomys chrysophilus and Aethomys ineptus and from Australia the fawn hopping mouse Notomys cervinus and the spinifex hopping mouse Notomys alexis . Cauda epididymal sperm smears were prepared and sperm midpiece and total tail lengths were determined. A linear mixed-effects model was used to estimate intra-individual variance. The results showed that in all three species where there are variable sperm head shapes ( B. indica , A. ineptus and N. alexis ), statistically significantly greater intra-individual variability of sperm midpiece and total tail lengths occurs ( P <0.0001 in all cases). These species all have relatively smaller testes mass compared with the closely related species with monomorphic sperm populations. This suggests that depressed levels of intermale sperm competition may result in the occurrence of variability in not only the divergent sperm head shape but also in the length of the midpiece as well as that of the total length of the sperm tail.     

Variability in sperm form and function in the context of sperm competition risk in two Tupinambis lizards

In polyandrous species, sperm morphometry and sperm velocity are under strong sexual selection. Although several hypotheses have been proposed to explain the role of sperm competition in sperm trait variation, this aspect is still poorly understood. It has been suggested that an increase in sperm competition pressure could reduce sperm size variation or produce a diversity of sperm to maximize male fertilization success. We aim at elucidating the variability of sperm morphometric traits and velocity in two Tupinambis lizards in the context of sperm competition risk. Sperm traits showed substantial variation at all levels examined: between species, among males within species, and within the ejaculate of individual males. Sperm velocity was found to be positively correlated with flagellum: midpiece ratio, with relatively longer flagella associated with faster sperm. Our results document high variability in sperm form and function in lizards.