Selection on sperm size in response to promiscuity and variation in female sperm storage organs.

Sperm cells are exceptionally morphologically diverse across taxa. However, morphology can be quite uniform within species, particularly for species where females copulate with many males per reproductive bout. Strong sexual selection in these promiscuous species is widely hypothesized to reduce intraspecific sperm variation. Conversely, we hypothesize that intraspecific sperm size variation may be maintained by high among-female variation in the size of sperm storage organs, assuming that paternity success improves when sperm are compatible in size with the sperm storage organ. We use individual-based simulations and an analytical model to evaluate how selection on sperm size depends on promiscuity level and variation in sperm storage organ size (hereafter, female preference variation). Simulations of high promiscuity (10 mates per female) showed stabilizing selection on sperm when female preference variation was low, and disruptive selection when female preference variation was high, consistent with the analytical model results. With low promiscuity (2-3 mates per female), selection on sperm was stabilizing for all levels of female preference variation in the simulations, contrasting with the analytical model. Promiscuity level, or mate sampling, thus has a strong impact on the selection resulting from female preferences. Furthermore, when promiscuity is low, disruptive selection on male traits will occur under much more limited circumstances (i.e. only with higher among-female variation) than many previous models suggest. Variation in female sperm storage organs likely has strong implications for intraspecific sperm variation in highly promiscuous species, but likely does not explain differences in intraspecific sperm variation for less promiscuous taxa.

Sperm morphology and performance in relation to postmating prezygotic isolation in two recently diverged passerine species.

Divergence in sperm phenotype and female reproductive environment may be a common source of postmating prezygotic (PMPZ) isolation between species. However, compared to other reproductive barriers it has received much less attention. In this study, we examined sperm morphology and velocity in two hybridizing passerine species, the common nightingale (Luscinia megarhynchos) and thrush nightingale (L. luscinia). In addition, we for the first time characterized a passerine female reproductive tract fluid proteome. We demonstrate that spermatozoa of the common nightingale have significantly longer and wider midpiece (proximal part of the flagellum containing mitochondria) and longer tail compared to spermatozoa of thrush nightingale. On the other hand, they have significantly shorter and narrower acrosome. Importantly, these differences did not have any effect on sperm velocity. Furthermore, the fluid from the reproductive tract of common nightingale females did not differentially affect velocity of conspecific and heterospecific sperm. Our results indicate that the observed changes in the flagellum and acrosome size are unlikely to contribute to PMPZ isolation through differential sperm velocity of conspecific and heterospecific sperm in the female reproductive tract. However, they could affect other postcopulatory processes, which might be involved in PMPZ isolation, such as sperm storage, longevity or sperm-egg interaction.

Flagellum tapering and midpiece volume in songbird spermatozoa.

In contrast to numerous studies on spermatozoa length, relatively little work focuses on the width of spermatozoa, and particularly the width of the midpiece and flagellum. In flagellated spermatozoa, the flagellum provides forward thrust while energy may be provided via mitochondria in the midpiece and/or through glycolysis along the flagellum itself. Longer flagella may be able to provide greater thrust but may also require stronger structural features and more or larger mitochondria to supply sufficient energy. Here, we use scanning electron microscopy to investigate the ultrastructure of spermatozoa from 55 passerine species in 26 taxonomic families in the Passerides infraorder. Our data confirm the qualitative observation that the flagellum tapers along its length, and we show that longer flagella are wider at the neck. This pattern is similar to mammals, and likely reflects the need for longer cells to be stronger against shearing forces. We further estimate the volume of the mitochondrial helix and show that it correlates well with midpiece length, supporting the use of midpiece length as a proxy for mitochondrial volume, at least in between-species studies where midpiece length is highly variable. These results provide important context for understanding the evolutionary correlations among different sperm cell components and dimensions.

Development and characterization of microsatellite primers for Triops granarius (Branchiopoda: Notostraca) using MiSeq technology.

BACKGROUND: Next-generation sequencing technology has allowed for the rapid development of microsatellites, neutral polymorphic markers that can be used for the analysis of population structure. METHODS AND RESULTS: In this study, we performed whole-genome sequencing using the Illumina MiSeq system and de novo assembly to design microsatellite primers for Triops granarius populations in Qatar. The developed microsatellites are suitable for future studies of genetic structuring among geographically isolated freshwater pools. A total of 23 different primer pairs produced typical microsatellite results, with each pair successfully amplified in up to 40 individuals. Only five of the loci produced a significant departure from Hardy-Weinberg equilibrium. CONCLUSIONS: Some of the underlying mechanisms regarding the few loci that deviated from HWE may be further investigated to determine the source of deviation. As T. granarius is the most widely distributed species of the family, the development of these molecular markers would be useful for conducting population genetics and biogeographical studies broadly.

Sperm length divergence as a potential prezygotic barrier in a passerine hybrid zone.

The saltmarsh sparrow Ammospiza caudacuta and Nelson's sparrow A. nelsoni differ in ecological niche, mating behavior, and plumage, but they hybridize where their breeding distributions overlap. In this advanced hybrid zone, past interbreeding and current backcrossing result in substantial genomic introgression in both directions, although few hybrids are currently produced in most locations. However, because both species are nonterritorial and have only brief male-female interactions, it is difficult to determine to what extent assortative mating explains the low frequency of hybrid offspring. Since females often copulate with multiple males, a role of sperm as a postcopulatory prezygotic barrier appears plausible. Here, we show that sperm length differs between the two species in the hybrid zone, with low among-male variation consistent with strong postcopulatory sexual selection on sperm cells. We hypothesize that divergence in sperm length may constitute a reproductive barrier between species, as sperm length co-evolves with the size of specialized female sperm storage tubules. Sperm does not appear to act as a postzygotic barrier, as sperm from hybrids was unexceptional.

Longer Sperm Swim More Slowly in the Canary Islands Chiffchaff.

Sperm swimming performance affects male fertilization success, particularly in species with high sperm competition. Understanding how sperm morphology impacts swimming performance is therefore important. Sperm swimming speed is hypothesized to increase with total sperm length, relative flagellum length (with the flagellum generating forward thrust), and relative midpiece length (as the midpiece contains the mitochondria). We tested these hypotheses and tested for divergence in sperm traits in five island populations of Canary Islands chiffchaff (Phylloscopus canariensis). We confirmed incipient mitochondrial DNA differentiation between Gran Canaria and the other islands. Sperm swimming speed correlated negatively with total sperm length, did not correlate with relative flagellum length, and correlated negatively with relative midpiece length (for Gran Canaria only). The proportion of motile cells increased with relative flagellum length on Gran Canaria only. Sperm morphology was similar across islands. We thus add to a growing number of studies on passerine birds that do not support sperm morphology-swimming speed hypotheses. We suggest that the swimming mechanics of passerine sperm are sufficiently different from mammalian sperm that predictions from mammalian hydrodynamic models should no longer be applied for this taxon. While both sperm morphology and sperm swimming speed are likely under selection in passerines, the relationship between them requires further elucidation.

Common Field Data Limitations Can Substantially Bias Sexual Selection Metrics.

Sexual selection studies widely estimate several metrics, but values may be inaccurate because standard field methods for studying wild populations produce limited data (e.g., incomplete sampling, inability to observe copulations directly). We compared four selection metrics (Bateman gradient, opportunity for sexual selection, opportunity for selection, and smax') estimated with simulated complete and simulated limited data for 15 socially monogamous songbird species with extrapair paternity (4%-54% extrapair offspring). Inferring copulation success from offspring parentage creates nonindependence between these variables and systematically underestimates copulation success. We found that this introduces substantial bias for the Bateman gradient, opportunity for sexual selection, and smax'. Notably, 47.5% of detected Bateman gradients were significantly positive for females, suggesting selection on females to copulate with multiple males, although the true Bateman gradient was zero. Bias generally increased with the extent of other sources of data limitations tested (nest predation, male infertility, and unsampled floater males). Incomplete offspring sampling introduced bias for all of the metrics except the Bateman gradient, while incomplete sampling of extrapair sires did not introduce additional bias when sires were a random subset of breeding males. Overall, our findings demonstrate how biases due to field data limitations can strongly impact the study of sexual selection.

Sperm divergence in a passerine contact zone: Indication of reinforcement at the gametic level.

Postcopulatory sexual selection may promote evolutionary diversification in sperm form, but the contribution of between-species divergence in sperm morphology to the origin of reproductive isolation and speciation remains little understood. To assess the possible role of sperm diversification in reproductive isolation, we studied sperm morphology in two closely related bird species, the common nightingale (Luscinia megarhynchos) and the thrush nightingale (Luscinia luscinia), that hybridize in a secondary contact zone spanning Central and Eastern Europe. We found: (1) striking divergence between the species in total sperm length, accompanied by a difference in the length of the mitochondrial sperm component; (2) greater divergence between species in sperm morphology in sympatry than in allopatry, with evidence for character displacement in sperm head length detected in L. megarhynchos; (3) interspecific hybrids showing sperm with a length intermediate between the parental species, but no evidence for decreased sperm quality (the proportion of abnormal spermatozoa in ejaculates). Our results demonstrate that divergence in sperm morphology between the two nightingale species does not result in intrinsic postzygotic isolation, but may contribute to postcopulatory prezygotic isolation. This isolation could be strengthened in sympatry by reinforcement.

Sperm morphology, sperm motility and paternity success in the bluethroat (Luscinia svecica).

Postcopulatory sexual selection may select for male primary sexual characteristics like sperm morphology and sperm motility, through sperm competition or cryptic female choice. However, how such characteristics influence male fertilization success remains poorly understood. In this study, we investigate possible correlations between sperm characteristics and paternity success in the socially monogamous bluethroat (Luscinia svecica svecica), predicting that sperm length and sperm swimming speed is positively correlated with paternity success. In total, 25% (15/61) of broods contained extra-pair offspring and 10% (33/315) of the offspring were sired by extra-pair males. Paternity success did not correlate significantly with sperm morphology or any aspects of sperm motility. Furthermore, sperm morphology and sperm motility did not correlate significantly with male morphological characters that previously have been shown to be associated with paternity success. Thus, the sperm characteristics investigated here do not appear to be strong predictors of paternity success in bluethroats.

Salinity-induced phenotypic plasticity in threespine stickleback sperm activation.

Phenotypic expression may be and often is influenced by an organism's developmental environment, referred to as phenotypic plasticity. The sperm cells of teleosts have been found to be inactive in the seminal plasma and are activated by osmotic shock for most fish species, through release in either hypertonic (for marine fish) or hypotonic (for freshwater fish) water. If this is the case, the regulatory system of sperm mobility should be reversed in salt- and freshwater fish. We tested this hypothesis by first activating sperm of salt- and freshwater populations of threespine stickleback in salt- and freshwater. The sperm from saltwater stickleback could be activated in either salinity, which matches the freshwater colonization history of the species, whereas the sperm from the freshwater population acted as predicted by the osmotic shock theory and was activated in freshwater only. As the freshwater population used here was calculated to be thousands of years old, we went on to test whether the trait(s) were plastic and sperm from freshwater males still could be activated in saltwater after individuals were exposed to saltwater. After raising freshwater stickleback in saltwater, we found the mature males to have active sperm in both saltwater and freshwater. Further, we also found the sperm of wild-caught freshwater stickleback to be active in saltwater after exposing those mature males to saltwater for only 2 days. This illustrates that the ability for stickleback sperm to be activated in a range of water qualities is an environmentally induced plastic trait.

Females discriminate against heterospecific sperm in a natural hybrid zone.

When hybridization is maladaptive, species-specific mate preferences are selectively favored, but low mate availability may constrain species-assortative pairing. Females paired to heterospecifics may then benefit by copulating with multiple males and subsequently favoring sperm of conspecifics. Whether such mechanisms for biasing paternity toward conspecifics act as important reproductive barriers in socially monogamous vertebrate species remains to be determined. We use a combination of long-term breeding records from a natural hybrid zone between collared and pied flycatchers (Ficedula albicollis and F. hypoleuca), and an in vitro experiment comparing conspecific and heterospecific sperm performance in female reproductive tract fluid, to evaluate the potential significance of female cryptic choice. We show that the females most at risk of hybridizing (pied flycatchers) frequently copulate with multiple males and are able to inhibit heterospecific sperm performance. The negative effect on heterospecific sperm performance was strongest in pied flycatcher females that were most likely to have been previously exposed to collared flycatcher sperm. We thus demonstrate that a reproductive barrier acts after copulation but before fertilization in a socially monogamous vertebrate. While the evolutionary history of this barrier is unknown, our results imply that there is opportunity for it to be accentuated via a reinforcement-like process.

Sperm performance in conspecific and heterospecific female fluid.

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.

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.

Morphology-function relationships and repeatability in the sperm of Passer sparrows.

Sperm performance is likely to be an important determinant of male reproductive success, especially when females copulate with multiple males. Understanding sperm performance is therefore crucial to fully understand the evolution of male reproductive strategies. In this study, we examined the repeatability of sperm morphology and motility measures over three breeding seasons, and we studied relationships between sperm morphology and function. We conducted this study in wild-derived captive house sparrows (Passer domesticus) and Spanish sparrows (P. hispaniolensis). Results for the two species were similar. As predicted from results in other passerine species, total sperm length was highly repeatable across ejaculates, and repeatability for the length of other components was moderate. The repeatability of sperm swimming speed across ejaculates was lower, but statistically significant, suggesting that sperm velocity may be a relatively dynamic trait. Surprisingly, swimming speed did not correlate with the relative length of the midpiece, and it correlated negatively with the relative length of the flagellum and with total sperm length. This pattern is the opposite of what theory predicts and differs from what has been found in house sparrows before. Also contrary to previous work, we found no evidence that total sperm length correlates with sperm longevity. These results therefore highlight the need for a better understanding of relationships between sperm morphology and function in passerine birds.

Physically challenging song traits, male quality, and reproductive success in house wrens.

Physically challenging signals are likely to honestly indicate signaler quality. In trilled bird song two physically challenging parameters are vocal deviation (the speed of sound frequency modulation) and trill consistency (how precisely syllables are repeated). As predicted, in several species, they correlate with male quality, are preferred by females, and/or function in male-male signaling. Species may experience different selective pressures on their songs, however; for instance, there may be opposing selection between song complexity and song performance difficulty, such that in species where song complexity is strongly selected, there may not be strong selection on performance-based traits. I tested whether vocal deviation and trill consistency are signals of male quality in house wrens (Troglodytes aedon), a species with complex song structure. Males' singing ability did not correlate with male quality, except that older males sang with higher trill consistency, and males with more consistent trills responded more aggressively to playback (although a previous study found no effect of stimulus trill consistency on males' responses to playback). Males singing more challenging songs did not gain in polygyny, extra-pair paternity, or annual reproductive success. Moreover, none of the standard male quality measures I investigated correlated with mating or reproductive success. I conclude that vocal deviation and trill consistency do not signal male quality in this species.

The deterrent effect of bird song in territory defense.

Using the responses of territory owners to playback to infer the territorial function of acoustic signals is common practice. However, difficulties with interpreting the results of such experiments have obscured our understanding of territorial signalling. For instance, a stronger response to playback is often interpreted as more aggressive, but there is no consensus as to whether this should be in response to the least or most threatening simulated intruder. Rather than following a gradual increase or decrease, the relationship between signal intensity and response strength may instead describe a peaked curve. We manipulated banded wren (Thryophilus pleurostictus) songs to simulate low-, median-, and high-performance singers and used these songs as stimuli in playback experiments. Banded wrens were less likely to approach the high-performance stimulus compared with the low- and median-performance stimuli. However, the birds that did approach the high-performance stimulus sang more than those that approached the low-performance stimulus. In addition, birds were more likely to match the songs when exposed to the median- and high-performance stimuli compared with the low-performance stimuli, and song matching predicted approach behavior. These results are in accordance with theoretical models of aggressive encounters in which low-performance opponents are challenged without further assessment. Median- and high-performance opponents, however, may require further assessment, and the latter may be perceived as too intimidating for approach.

Isolation and characterization of SNP variation at 90 anonymous loci in the banded wren (Thryothorus pleurostictus).

Single nucleotide polymorphisms (SNPs) are becoming more commonly used as molecular markers in conservation studies. However, relatively few studies have employed SNPs for species with little or no existing sequence data, partly due to the practical challenge of locating appropriate SNP loci in these species. Here we describe an application of SNP discovery via shotgun cloning that requires no pre-existing sequence data and is readily applied to all taxa. Using this method, we isolated, cloned and screened for SNP variation at 90 anonymous sequence loci (51kb total) from the banded wren (Thryothorus pleurostictus), a Central American species with minimal pre-existing sequence data. We identified 168 SNPs (a mean of one SNP/305 bp, with SNPs unevenly distributed across loci). Further characterization of variation at 41 of these SNP loci among 256 individuals including 37 parent-offspring families suggests that they provide substantial information for defining the genetic mating system of this species, and that SNPs may be generally useful for this purpose when other markers are problematic.