Sperm Numbers as a Paternity Guard in a Wild Bird

Sperm competition is thought to impose strong selection on males to produce competitive ejaculates to outcompete rival males under competitive mating conditions. Our understanding of how different sperm traits influence fertilization success, however, remains limited, especially in wild populations. Recent literature highlights the importance of incorporating multiple ejaculate traits and pre-copulatory sexually selected traits in analyses aimed at understanding how selection acts on sperm traits. However, variation in a male’s ability to gain fertilization success may also depend upon a range of social and ecological factors that determine the opportunity for mating events both within and outside of the social pair-bond. Here, we test for an effect of sperm quantity and sperm size on male reproductive success in the red-back fairy-wren (Malurus melanocephalus) while simultaneously accounting for pre-copulatory sexual selection and potential socio-ecological correlates of male mating success. We found that sperm number (i.e., cloacal protuberance volume), but not sperm morphology, was associated with reproductive success in male red-backed fairy-wrens. Most notably, males with large numbers of sperm available for copulation achieved greater within-pair paternity success. Our results suggest that males use large sperm numbers as a defensive strategy to guard within-pair paternity success in a system where there is a high risk of sperm competition and female control of copulation. Finally, our work highlights the importance of accounting for socio-ecological factors that may influence male mating opportunities when examining the role of sperm traits in determining male reproductive success.

Chronic Background Radiation Correlates With Sperm Swimming Endurance in Bank Voles From Chernobyl

Sperm quantity and quality are key features explaining intra- and interspecific variation in male reproductive success. Spermatogenesis is sensitive to ionizing radiation and laboratory studies investigating acute effects of ionizing radiation have indeed found negative effects of radiation on sperm quantity and quality. In nature, levels of natural background radiation vary dramatically, and chronic effects of low-level background radiation exposure on spermatogenesis are poorly understood. The Chernobyl region offers a unique research opportunity for investigating effects of chronic low-level ionizing radiation on reproductive properties of wild organisms. We captured male bank voles (Myodes glareolus) from 24 locations in the Chernobyl exclusion zone in 2011 and 2015 and collected information on sperm morphology and kinetics. The dataset is limited in size and there overall was a relatively weak correlation between background radiation and sperm quality. Still, some correlations are worth discussing. First, mid-piece segments of spermatozoa tended to be smaller in bank vole males from areas with elevated background radiation levels. Second, we demonstrated a significant positive relationship between background radiation dose rates and the proportion of static spermatozoa among males within and among study locations after 10 as well as 60 min of incubation. Our results provide novel evidence of damaging effects of low dose ionizing radiation on sperm performance in wild rodent populations, and highlight that this topic requires further study across the natural gradients of background radiation that exist in nature.

Recurrent co-option and recombination of cytokine and three finger proteins in multiple reproductive tissues throughout salamander evolution

The proteomic composition of amphibian gametes is largely a molecular mystery, particularly for Urodeles (salamanders and newts) which have few genomic-scale resources. Lungless salamanders (family Plethodontidae) include approximately two thirds of all extant salamander species and are classic models of vertebrate mating behavior. As part of an extended, multi-stage courtship ritual, male plethodontid salamanders deliver rapidly evolving protein pheromones that modify female behavior and improve male reproductive success. Despite great interest in this set of pre-mating reproductive barriers, limited characterization of plethodontid gametes has prohibited investigation of post-mating pre-zygotic barriers such as sperm-egg recognition. In this study, we performed transcriptomic analyses of testis and ovary using long-read PacBio sequencing and proteomic analyses of sperm using mass spectrometry for two evolutionary divergent plethodontid species, Plethodon shermani and Desmognathus ocoee. In both species, many of the most abundant sperm proteins were paralogs of the courtship pheromones Plethodontid Receptivity Factor (PRF), Plethodontid Modulating Factor (PMF), and Sodefrin Precursor-like Factor (SPF). Sperm-specific paralogs of PMF and SPF are likely the most abundant secreted proteins in P. shermani and D. ocoee, respectively. In contrast, sperm PRF lacks a signal peptide and may be expressed in cytoplasm. PRF pheromone genes evolved independently multiple times through repeated gene duplication of sperm PRF genes and signal peptides recovered by recombination with PMF genes. Phylogenetic analysis of courtship pheromones and their sperm paralogs support that each protein family evolved for these two reproductive contexts at distinct evolutionary time points between 17 and 360 million years ago. As the first molecular characterization of salamander gametes, this study expands our knowledge of amphibian fertilization beyond frogs and provides novel insight into the evolutionary processes by which new, rapidly evolving reproductive proteins may evolve.

Disentangling the determinants of reproductive success in a temperate reef fish, Forsterygion lapillum

<p>Identifying sources of individual variation in reproductive success has been a longstanding challenge for evolutionary ecologists. Reproductive success among individuals can be due to several factors such as competition between conspecifics for nest sites and mating partners, mate choice, or by the physical environment. Reproductive success, particularly among males, can be extremely diverse both within and between species and determining which components contribute to success can be particularly challenging. In this thesis, I investigated patterns and drivers of reproductive success in a temperate marine reef fish, Forsterygion lapillum (the common triplefin). Specifically, I examined how male quality, nest quality, and female choice influence male reproductive success. Additionally, I quantified male reproductive success during the winter and summer of the breeding season to examine the temporal dynamics of breeding success in F. lapillum. Selection of mates by females can be driven by the quality and behavioural attributes of the male or by the quality of resources offered. In Chapter 2, using field-based observations, combined with a lab-based study, I evaluated the effects of different male traits and nest characteristics on female choice and male reproductive success. Specifically, I observed egg guarding males in the field during the breeding season and recorded their phenotypic traits, behaviours, and nest characteristics. I then examined their influence on 3 different components of male reproductive success (brood size, individual egg size, and mate attraction). Additionally, I conducted dichotomous choice tests in the laboratory to evaluate female preference for different sized males, holding different sized nests. In the field, I did not detect a significant relationship between male mating success and male total length or nest size. Brood size and individual egg size were highly variable among sampled males, however, further factors such as courtship frequency, and the number of interactions with potential predators did not explain any additional variation. The number of agonistic displays performed by egg guarding males was the only factor to influence egg size, however, it had no direct impact on brood size or mate attraction. On the contrary, results from the laboratory experiment suggested that male total length and nest size were important during female choice. Females were attracted to and spawned more frequently with larger males holding larger nests. Additionally, females showed a particular preference towards males that displayed intense courtship behaviours. These results suggest that variation in reproductive success among individuals is not random in the common triplefin (F. lapillum) and may be due to a range of complex factors. In natural systems, individual variation in mating success is known to be highly dynamic and vary over time. In Chapter 3, I addressed 3 questions related to reproductive success in male common triplefin: 1) Does the operational sex ratio (OSR) and the density of individuals change predictably within the breeding season? 2) Does male reproductive success change within the breeding season? And 3) Does the age and growth rate of successful males change within the breeding season? To address these questions, I sampled a population of F. lapillum during two periods of the breeding season and quantified a set of morphological and physical traits. Furthermore, I reconstructed individual life histories from the otoliths of egg guarding males. My results show that the density of individuals in the population increased during the summer months, but the operational sex ratio (OSR) remained male-biased. Male reproductive success in terms of brood size and average egg size did not fluctuate during the sampling period. However, the size of males and the size of the nest (cobblestone) held by males was significantly larger in summer compared to winter. Interestingly, successful males sampled in the winter had hatched significantly earlier than successful males sampled in the summer, but their average growth rate remained similar. These findings indicate that variation in male traits across the breeding season plays an important role in female mate choice. The mating system and pool of mating individuals in the common triplefin (F. lapillum) is highly dynamic over the year and has the potential to shape the success of individuals. Overall, this study emphasizes the importance of considering multiple cues and temporal dynamics when disentangling the determinants of individual reproductive success. These findings suggest that male-male competition and female mate choice have a significant influence on male reproductive success. The reproductive ecology of F. lapillum is highly complex and my research has provided valuable insight into its dynamic nature. These results may apply to other species with male parental care and provides an important contribution towards understanding sexual selection and the evolution of mating systems with male parental care.</p>

Disentangling the determinants of reproductive success in a temperate reef fish, Forsterygion lapillum

<p>Identifying sources of individual variation in reproductive success has been a longstanding challenge for evolutionary ecologists. Reproductive success among individuals can be due to several factors such as competition between conspecifics for nest sites and mating partners, mate choice, or by the physical environment. Reproductive success, particularly among males, can be extremely diverse both within and between species and determining which components contribute to success can be particularly challenging. In this thesis, I investigated patterns and drivers of reproductive success in a temperate marine reef fish, Forsterygion lapillum (the common triplefin). Specifically, I examined how male quality, nest quality, and female choice influence male reproductive success. Additionally, I quantified male reproductive success during the winter and summer of the breeding season to examine the temporal dynamics of breeding success in F. lapillum. Selection of mates by females can be driven by the quality and behavioural attributes of the male or by the quality of resources offered. In Chapter 2, using field-based observations, combined with a lab-based study, I evaluated the effects of different male traits and nest characteristics on female choice and male reproductive success. Specifically, I observed egg guarding males in the field during the breeding season and recorded their phenotypic traits, behaviours, and nest characteristics. I then examined their influence on 3 different components of male reproductive success (brood size, individual egg size, and mate attraction). Additionally, I conducted dichotomous choice tests in the laboratory to evaluate female preference for different sized males, holding different sized nests. In the field, I did not detect a significant relationship between male mating success and male total length or nest size. Brood size and individual egg size were highly variable among sampled males, however, further factors such as courtship frequency, and the number of interactions with potential predators did not explain any additional variation. The number of agonistic displays performed by egg guarding males was the only factor to influence egg size, however, it had no direct impact on brood size or mate attraction. On the contrary, results from the laboratory experiment suggested that male total length and nest size were important during female choice. Females were attracted to and spawned more frequently with larger males holding larger nests. Additionally, females showed a particular preference towards males that displayed intense courtship behaviours. These results suggest that variation in reproductive success among individuals is not random in the common triplefin (F. lapillum) and may be due to a range of complex factors. In natural systems, individual variation in mating success is known to be highly dynamic and vary over time. In Chapter 3, I addressed 3 questions related to reproductive success in male common triplefin: 1) Does the operational sex ratio (OSR) and the density of individuals change predictably within the breeding season? 2) Does male reproductive success change within the breeding season? And 3) Does the age and growth rate of successful males change within the breeding season? To address these questions, I sampled a population of F. lapillum during two periods of the breeding season and quantified a set of morphological and physical traits. Furthermore, I reconstructed individual life histories from the otoliths of egg guarding males. My results show that the density of individuals in the population increased during the summer months, but the operational sex ratio (OSR) remained male-biased. Male reproductive success in terms of brood size and average egg size did not fluctuate during the sampling period. However, the size of males and the size of the nest (cobblestone) held by males was significantly larger in summer compared to winter. Interestingly, successful males sampled in the winter had hatched significantly earlier than successful males sampled in the summer, but their average growth rate remained similar. These findings indicate that variation in male traits across the breeding season plays an important role in female mate choice. The mating system and pool of mating individuals in the common triplefin (F. lapillum) is highly dynamic over the year and has the potential to shape the success of individuals. Overall, this study emphasizes the importance of considering multiple cues and temporal dynamics when disentangling the determinants of individual reproductive success. These findings suggest that male-male competition and female mate choice have a significant influence on male reproductive success. The reproductive ecology of F. lapillum is highly complex and my research has provided valuable insight into its dynamic nature. These results may apply to other species with male parental care and provides an important contribution towards understanding sexual selection and the evolution of mating systems with male parental care.</p>

Cohesion in male singing behavior predicts group reproductive output in a social songbird

All social groups require organization to function optimally. Group organization is often shaped by social 'rules', which function to manage conflict, discourage cheating, or promote cooperation. If social rules promote effective social living, then the ability to learn and follow these rules may be expected to influence individual and group-level fitness. However, such links can rarely be tested, due to the complexity of the factors mediating social systems and the difficulty of gathering data across multiple groups. Songbirds offer an opportunity to investigate the link between social rules and reproductive output because most of their social interactions are mediated by song, a well-studied and readily quantifiable behavior. Using observations from 19 groups of brown-headed cowbirds (Molothrus ater) studied across 15 years, we find evidence for a previously undocumented social rule: cohesive group transitions between dominance- and courtship-related singing. Comparing across groups, the degree of cohesion in male singing behavior predicts the reproductive output of their group. Experimental manipulation of group structure via the introduction of juvenile males to captive flocks reduced group cohesion and adult male reproductive success. Taken together, these results demonstrate that cohesion in group behavioral states can affect both individual and group-level reproductive success, suggesting that selection can act not only on individual-level traits, but also on an individual's ability and opportunity to participate effectively in organized social interactions. Social cohesion could therefore be an unappreciated force affecting social evolution in many diverse systems.

Genetic coordination of sperm morphology and seminal fluid proteins promotes male reproductive success in Drosophila melanogaster

Spermatozoal morphology is highly variable both among and within species and in ways that can significantly impact fertilization success. In Drosophila melanogaster, paternity success depends on sperm length of both competing males and length of the female's primary sperm storage organ. We found that genes upregulated in long sperm testes are enriched for lncRNAs and seminal fluid proteins (Sfps). Transferred in seminal fluid to the female during mating, Sfps are secreted by the male accessory glands (AG) and affect female remating rate, physiology, and behavior with concomitant advantages for male reproductive success. Despite being upregulated in long sperm testes, they have no known function in testis tissue. We found that Sex Peptide and ovulin (Acp26Aa) knockouts resulted in shorter sperm, suggesting that Sfps may regulate sperm length during spermatogenesis. However, knockout of AG function did not affect sperm length, suggesting that AG expression has no influence on spermatogenic processes. We also found that long sperm males are better able to delay female remating, suggesting higher Sfp expression in AG. These results might suggest that long sperm males have a double advantage in sperm competition by both delaying female remating, likely through transfer of more Sfps, and by resisting sperm displacement. However, we also found that this extra advantage does not necessarily translate to more progeny or higher paternity success. Thus, we found that multiple components of the ejaculate coordinate to promote male reproductive success at different stages of reproduction, but the realized fitness advantages in sperm competition are uncertain.

Pheromones that correlate with reproductive success in competitive conditions

The major urinary proteins (MUPs) of house mice (Mus musculus) bind and stabilize the release of pheromones and other volatile organic compounds (VOCs) from urinary scent marks, which mediate chemical communication. Social status influences MUP and VOC excretion, and the urinary scent of dominant males is attractive to females. Urinary pheromones influence the sexual behavior and physiology of conspecifics, and yet it is not known whether they also affect reproductive success. We monitored the excretion of urinary protein and VOCs of wild-derived house mice living in large seminatural enclosures to compare the sexes and to test how these compounds correlate with reproductive success. Among males, urinary protein concentration and VOC expression correlated with reproductive success and social status. Territorial dominance also correlated with reproductive success in both sexes; but among females, no urinary compounds were found to correlate with social status or reproductive success. We found several differences in the urinary protein and volatile pheromones of mice in standard cages versus seminatural enclosures, which raises caveats for conventional laboratory studies. These findings provide novel evidence for chemical signals that correlate with male reproductive success of house mice living in competitive conditions.

Selection on Sperm Count, but Not on Sperm Morphology or Velocity, in a Wild Population of Anolis Lizards

Sperm competition is a widespread phenomenon that shapes male reproductive success. Ejaculates present many potential targets for postcopulatory selection (e.g., sperm morphology, count, and velocity), which are often highly correlated and potentially subject to complex multivariate selection. Although multivariate selection on ejaculate traits has been observed in laboratory experiments, it is unclear whether selection is similarly complex in wild populations, where individuals mate frequently over longer periods of time. We measured univariate and multivariate selection on sperm morphology, sperm count, and sperm velocity in a wild population of brown anole lizards (Anolis sagrei). We conducted a mark-recapture study with genetic parentage assignment to estimate individual reproductive success. We found significant negative directional selection and negative quadratic selection on sperm count, but we did not detect directional or quadratic selection on any other sperm traits, nor did we detect correlational selection on any trait combinations. Our results may reflect pressure on males to produce many small ejaculates and mate frequently over a six-month reproductive season. This study is the first to measure multivariate selection on sperm traits in a wild population and provides an interesting contrast to experimental studies of external fertilizers, which have found complex multivariate selection on sperm phenotypes.

Linking Male Reproductive Success to Effort Within and Among Nests in a Co-Breeding Stream Fish

Nest construction is an energetically costly behavior displayed by males in many taxa. In some species, males construct nests and co-breed with other males and they may construct multiple nests in a breeding season. However, little is understood about how allocation of effort within and among nests affects male reproductive success. We characterized reproductive effort of male bluehead chub (Nocomis leptocephalus) on nests in an entire breeding season using PIT antennas deployed around nests and linked effort within and among nests to reproductive success, measured by number of offspring assigned genetically to each male, in a small stream in South Carolina, USA. We monitored time spent by a total of 34 males on each of 18 nests during the spawning season in 2017. A Bayesian hierarchical analysis showed that larger males spent more time constructing and maintaining a given nest, and consequently were more reproductively successful than smaller males on the same nest. Combined with aggressive behavior displayed by larger males toward smaller males, this finding suggested that reproductive effort including agonistic interactions within nests was a determinant of reproductive success. In contrast, more males together constructed larger nests, which led to higher reproductive success of members that constructed those nests. Number of nests that male constructed, a measure of effort across nests, was not a predictor of reproductive success, indicating that reproductive success varied among nests due to nest size. Our study showed that male reproductive success was determined by both aggressive and cooperative behaviors in a co-breeding species.