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Phillips, P. C.

Publications and source records attributed to Phillips, P. C..

At least 19 recordsLinked to original sources

Disruption of the insulin signaling pathway in C. elegans dramatically increases male longevity and enhances reproductive health late in life

Males and females are known to have dramatically different health and lifespan trajectories, but the underlying basis for these differences is only now being fully investigated1. In the Caenorhabditis elegans nematode model system, most aging studies have been conducted with hermaphrodites, and little is known about male-specific responses to pro-longevity mutations. Several previous studies have used the auxin-inducible degron system to degrade the insulin-like DAF-2/IGF-1 receptor in hermaphrodites, finding that both ubiquitous and tissue-specific degradation can extend lifespan2-4. Here we show that ubiquitous degradation of DAF-2 in male C. elegans increases median lifespan by more than 440%, one of the longest lifespan extensions by a single intervention to date. Conversely, degrading DAF-2 in the male germline decreased lifespan, opposite of its effect in hermaphrodites3. Using male mating and reproductive success as a meaningful ecological and neurophysiological measure of healthspan, we found that ubiquitous degradation of DAF-2 greatly prolongs reproductive health, likely by prolonging function of the male intromittent organ in the tail. This work highlights the importance of studying sex differences in aging and highlights the utility of using C. elegans males to understand the underlying basis of enhanced lifespan and healthspan.

genetics↗

Pro-longevity compounds extend Caenorhabditis elegans male lifespan and reproductive healthspan

Sex differences in aging are robust and ubiquitous. Demographic differences in aging generated by sex have long been recognized, but the underlying biological basis for these differences and the potential for sex-specific interventions remain understudied. To explore sex differences in the response to pro-longevity interventions, we utilized the C. elegans aging model and asked whether male lifespan and reproductive healthspan can be extended via compounds known to have pro-longevity effects in hermaphrodites. We tested seven different compounds at two concentrations each and found that lifespan was extended under all tested conditions. However, reproductive healthspan measured by mating success in late life improved under only two tested conditions, sulforaphane and metformin. These results demonstrate that lifespan and healthspan can be decoupled in C. elegans males and offer a new framework for screening pro-longevity compounds and for studying sex differences in aging in a classical aging model.

physiology↗

The broccoli derivative sulforaphane extends lifespan by slowing the transcriptional aging clock

Sulforaphane, an organosulfur isothiocyanate derived from cruciferous vegetables, has been shown to inhibit inflammation, oxidative stress, and cancer cell growth. To explore the potential of sulforaphane as a candidate natural compound for promoting longevity more generally, we tested the dose and age-specific effects of sulforaphane on C. elegans longevity, finding that it can extend lifespan by more than 50% at the most efficacious doses, but that treatment must be initiated early in life to be effective. We then created a novel, gene-specific, transcriptional aging clock, which demonstrated that sulforaphane-treated individuals exhibited a "transcriptional age" that was approximately four days younger than age-matched controls, representing a nearly 20% reduction in biological age. The clearest transcriptional responses were detoxification pathways, which, together with the shape of the dose-response curve, indicates a likely hormetic response to sulforaphane. These results support the idea that robust longevity-extending interventions can act via global effects across the organism, as revealed by systems level changes in gene expression.

physiology↗

Precise Lineage Tracking Using Molecular Barcodes Demonstrates Fitness Trade-offs for Ivermectin Resistance in Nematodes

A fundamental tenet of evolutionary genetics is that the direction and strength of selection on individual loci varies with the environment. Barcoded evolutionary lineage tracking is a powerful approach for high-throughput measurement of selection within experimental evolution that to date has largely been restricted to studies within microbial systems, largely because the random integration of barcodes within animals is limited by physical and molecular protection of the germline. Here, we use the recently developed TARDIS barcoding system in Caenorhabditis elegans (Stevenson et al., 2023) to implement the first randomly inserted genomic-barcode experimental evolution animal model and use this system to precisely measure the influence of the concentration of the anthelmintic compound ivermectin on the strength of selection on an ivermectin resistance cassette. The combination of the trio of knockouts in neuronally expressed GluCl channels, avr-14, avr-15, and glc-1, has been previously demonstrated to provide resistance to ivermectin at high concentrations. Varying the concentration of ivermectin in liquid culture allows the strength of selection on these genes to be precisely controlled within populations of millions of individuals, yielding the largest animal experimental evolution study to date. The frequency of each barcode was determined at multiple time points via sequencing at deep coverage and then used to estimate the fitness of the individual lineages in the population. The mutations display a high cost to resistance at low concentrations, rapidly losing out to wildtype genotypes, but the balance tips in their favor when the ivermectin concentration exceeds 2nM. This trade-off in resistance is likely generated by a hindered rate of development in resistant individuals. Our results demonstrate that C. elegans can be used to generate high precision estimates of fitness using a high-throughput barcoding approach to yield novel insights into evolutionarily and economically important traits.

evolutionary biology↗

Computer prediction and genetic analysis identifies retinoic acid modulation as a driver of conserved longevity pathways in genetically-diverse Caenorhabditis nematodes

Aging is a pan-metazoan process with significant consequences for human health and society--discovery of new compounds that ameliorate the negative health impacts of aging promise to be of tremendous benefit across a number of age-based comorbidities. One method to prioritize a testable subset of the nearly infinite universe of potential compounds is to use computational prediction of their likely anti-aging capacity. Here we present a survey of longevity effects for 16 compounds suggested by a previously published computational prediction set, capitalizing upon the comprehensive, multi-species approach utilized by the Caenorhabditis Intervention Testing Program (CITP). While eleven compounds (aldosterone, arecoline, bortezomib, dasatinib, decitabine, dexamethasone, erlotinib, everolimus, gefitinib, temsirolimus, and thalidomide) either had no effect on median lifespan or were toxic, five compounds (all-trans retinoic acid, berberine, fisetin, propranolol, and ritonavir) extended lifespan in Caenorhabditis elegans. These computer predictions yield a remarkable positive hit rate of 30%. Deeper genetic characterization of the longevity effects of one of the most efficacious compounds, the endogenous signaling ligand all-trans retinoic acid (atRA, designated tretinoin in medical products), which is widely prescribed for treatment of acne, skin photoaging and acute promyelocytic leukemia, demonstrated a requirement for the regulatory kinases AKT-1 and AKT-2. While the canonical Akt-target FOXO/DAF-16 was largely dispensable, other conserved Akt-targets (Nrf2/SKN-1 and HSF1/HSF-1), as well as the conserved catalytic subunit of AMPK AAK-2, were all necessary for longevity extension by atRA. Evolutionary conservation of retinoic acid as a signaling ligand and the structure of the downstream effector network of retinoic acid combine to suggest that the all-trans retinoic acid pathway is an ancient metabolic regulatory system that can modulate lifespan. Our results highlight the potential of combining computational prediction of longevity interventions with the power of nematode functional genetics and underscore that the manipulation of a conserved metabolic regulatory circuit by co-opting endogenous signaling molecules is a powerful approach for discovering aging interventions.

genetics↗

Pervasive conservation of intron number and other genetic elements revealed by a chromosome-level genomic assembly of the hyper-polymorphic nematode Caenorhabditis brenneri

With within-species genetic diversity estimates that span the gambit of that seen across the entirety of animals, the Caenorhabditis genus of nematodes holds unique potential to provide insights into how population size and reproductive strategies influence gene and genome organization and evolution. Our study focuses on Caenorhabditis brenneri, currently known as one of the most genetically diverse nematodes within its genus and metazoan phyla. Here, we present a high-quality gapless genome assembly and annotation for C. brenneri, revealing a common nematode chromosome arrangement characterized by gene-dense central regions and repeat rich peripheral parts. Comparison of C. brenneri with other nematodes from the Elegans group revealed conserved macrosynteny but a lack of microsynteny, characterized by frequent rearrangements and low correlation iof orthogroup sizes, indicative of high rates of gene turnover. We also assessed genome organization within corresponding syntenic blocks in selfing and outcrossing species, affirming that selfing species predominantly experience loss of both genes and intergenic DNA. Comparison of gene structures revealed strikingly small number of shared introns across species, yet consistent distributions of intron number and length, regardless of population size or reproductive mode, suggesting that their evolutionary dynamics are primarily reflective of functional constraints. Our study provides valuable insights into genome evolution and expands the nematode genome resources with the highly genetically diverse C. brenneri, facilitating research into various aspects of nematode biology and evolutionary processes.

genomics↗

Widespread changes in gene expression accompany body size evolution in nematodes

Body size is a fundamental trait that drives multiple evolutionary and ecological patterns. Caenorhabditis inopinata is a fig-associated nematode that is exceptionally large relative to other members of the genus, including C. elegans. We previously showed that C. inopinata is large primarily due to postembryonic cell size expansion that occurs during the larval-to-adult transition. Here, we describe gene expression patterns in C. elegans and C. inopinata throughout this developmental period to understand the transcriptional basis of body size change. We performed RNA-seq in both species across the L3, L4, and adult stages. Most genes are differentially expressed across all developmental stages, consistent with C. inopinatas divergent ecology and morphology. We also used a model comparison approach to identify orthologs with divergent dynamics across this developmental period between the two species. This included genes connected to neurons, behavior, stress response, developmental timing, and small RNA/chromatin regulation. Multiple hypodermal collagens were also observed to harbor divergent developmental dynamics across this period, and genes important for molting and body morphology were also detected. Genes associated with TGF-{beta} signaling revealed idiosyncratic and unexpected transcriptional patterns given their role in body size regulation in C. elegans. Widespread transcriptional divergence between these species is unexpected and may be a signature of the ecological and morphological divergence of C. inopinata. Alternatively, transcriptional turnover may be the rule in the Caenorhabditis genus, indicative of widespread developmental system drift among species. This work lays the foundation for future functional genetic studies interrogating the bases of body size evolution in this group.

genomics↗

Epigenetic context predicts gene expression variation and reproductive traits across genetically identical individuals

In recent decades, genome-wide association studies (GWAS) have been the major approach to understand the biological basis of individual differences in traits and diseases. However, GWAS approaches have limited predictive power to explain individual differences, particularly for complex traits and diseases in which environmental factors play a substantial role in their etiology. Indeed, individual differences persist even in genetically identical individuals, although fully separating genetic and environmental causation is difficult in most organisms. To understand the basis of individual differences in the absence of genetic differences, we measured two quantitative reproductive traits in 180 genetically identical young adult Caenorhabditis elegans roundworms in a shared environment and performed single-individual transcriptomics on each worm. We identified hundreds of genes for which expression variation was strongly associated with reproductive traits, some of which depended on individuals historical environments and some of which was random. Multiple small sets of genes together were highly predictive of reproductive traits, explaining on average over half and over a quarter of variation in the two traits. We manipulated mRNA levels of predictive genes to identify a set of causal genes, demonstrating the utility of this approach for both prediction and understanding underlying biology. Finally, we found that the chromatin environment of predictive genes was enriched for H3K27 trimethylation, suggesting that gene expression variation may be driven in part by chromatin structure. Together, this work shows that individual, non-genetic differences in gene expression are both highly predictive and causal in shaping reproductive traits.

genomics↗

The Egg-Counter: A novel microfluidic platform for characterization of Caenorhabditis elegans egg-laying

Reproduction is a fundamental process that shapes the demography of every living organism yet is often difficult to assess with high precision in animals that produce large numbers of offspring. Here, we present a novel microfluidic research platform for studying Caenorhabditis elegans egg-laying. The platform provides higher throughput than traditional solid-media assays while providing a very high degree of temporal resolution. Additionally, the environmental control enabled by microfluidic animal husbandry allows for experimental perturbations difficult to achieve with solid-media assays. We demonstrate the platforms utility by characterizing C. elegans egg-laying behavior at two commonly used temperatures, 15 and 20{degrees}C. As expected, we observed a delayed onset of egg-laying at 15{degrees}C degrees, consistent with published temperature effects on development rate. Additionally, as seen in solid media studies, egg laying output was higher under the canonical 20{degrees}C conditions. While we validated the Egg-Counter with a study of temperature effects in wild-type animals, the platform is highly adaptable to any nematode egg-laying research where throughput or environmental control needs to be maximized without sacrificing temporal resolution.

animal behavior and cognition↗

Post-insemination sexual selection in males indirectly masculinizes the female transcriptome

Sex-specific regulation of gene expression is the most plausible way for generating sexually differentiated phenotypes from an essentially shared genome. However, since genetic material is shared, sex-specific selection in one sex can have an indirect response in the other sex. From a gene expression perspective, this tethered response can move one sex away from their wildtype expression state and impact potentially many gene regulatory networks. Here, using experimental evolution in the model nematode Caenorhabditis elegans, we explore the coupling of direct sexual selection on males with the transcriptomic response in males and females over microevolutionary timescales to uncover the extent to which post-insemination reproductive traits share a genetic basis between the sexes. We find that differential gene expression evolved in a sex-specific manner in males, while in females indirect selection causes an evolved response. Almost all differentially expressed genes were downregulated in both evolved males and females. Moreover, 97% of significantly differentially expressed genes in males and 69% of significantly differentially expressed genes in females have wildtype female-biased expression profile. Changes in gene expression profiles were likely driven through trans-acting pathways that are shared between the sexes. We found no evidence that the core dosage compensation machinery was impacted by experimental evolution. Together these data suggest a de-feminization of the male transcriptome and masculinization of the female transcriptome driven by direct selection on male sperm competitive ability. Our results indicate that on short evolutionary timescales sexual selection can generate putative sexual conflict in expression space.

evolutionary biology↗

Heritable epigenetic variation facilitates long-term maintenance of epigenetic and genetic variation

The maintenance of genetic and phenotypic variation has long been one of the fundamental questions in population and quantitative genetics. A variety of factors have been implicated to explain the maintenance of genetic variation in some contexts (e.g. balancing selection), but the potential role of epigenetic regulation to influence population dynamics has been understudied. It is well recognized that epigenetic regulation, including histone methylation, small RNA expression, and DNA methylation, helps to define differences between cell types and facilitate phenotypic plasticity. In recent years, empirical studies have shown the potential for epigenetic regulation to also be heritable for at least a few generations without selection, raising the possibility that differences in epigenetic regulation can act alongside genetic variation to shape evolutionary trajectories. Like genetic mutation, heritable differences in epigenetic regulation can arise spontaneously; these are termed epimutations. Epimutations differ from genetic mutations in two key ways - they occur at a higher rate and the loci at which they occur often revert back to their original state within a few generations. Here, we present an extension of the standard population-genetic model with selection to incorporate epigenetic variation arising via epimutation. Our model assumes a diploid, sexually reproducing population with random mating. In addition to spontaneous genetic mutation, we included parameters for spontaneous epimutation and back-epimutation, allowing for four potential epialleles at a single locus (two genetic alleles, each with two epigenetic states), each of which affect fitness. We then analyzed the conditions under which stable epialleles were maintained. Our results show that highly reversible epialleles can be maintained in long-term equilibrium under neutral conditions in a manner that depends on the epimutation and back-epimutation rates, which we term epimutation-back-epimutation equilibrium. On the other hand, epialleles that compensate for deleterious mutations cause deviations from the expectations of mutation-selection balance by a simple factor that depends on the epimutation and back-epimutation rate. We also numerically analyze several sets of fitness parameters for which large deviations from mutation-selection balance occur. Together, these results demonstrate that transient epigenetic regulation may be an important factor in the maintenance of both epigenetic and genetic variation in populations.

evolutionary biology↗

Genetic diversity landscapes in outcrossing and selfing Caenorhabditis nematodes

Caenorhabditis nematodes form an excellent model for studying how the mode of reproduction affects genetic diversity, as some species reproduce via outcrossing whereas others can self-fertilize. Currently, chromosome-level patterns of diversity and recombination are only available for self-reproducing Caenorhabditis, making the generality of genomic patterns across the genus unclear given the profound potential influence of reproductive mode. Here we present a whole-genome diversity landscape, coupled with a new genetic map, for the outcrossing nematode C. remanei. We demonstrate that the genomic distribution of recombination in C. remanei, like the model nematode C. elegans, shows high recombination rates on chromosome arms and low rates toward the central regions. Patterns of genetic variation across the genome are also similar between these species, but differ dramatically in scale, being 10-fold greater for C. remanei. Historical reconstructions of variation in effective population size over the past million generations echo this difference in polymorphism. Evolutionary simulations demonstrate how selection, recombination, mutation, and selfing shape variation along the genome, and that multiple drivers can produce patterns similar to those observed in natural populations. Convolutional neural networks demonstrate the potential for classifying distinct evolutionary scenarios in simulated populations. Distinguishing these forces confidently with the empirical data, however, will benefit from larger population genomic samples from multiple populations and consideration of an even more extensive training set of simulations.

evolutionary biology↗

High-Throughput Library Transgenesis in Caenorhabditis elegans via Transgenic Arrays Resulting in Diversity of Integrated Sequences (TARDIS)

High-throughput transgenesis using synthetic DNA libraries is a powerful method for systematically exploring genetic function. Diverse synthesized libraries have been used for protein engineering, identification of protein-protein interactions, characterization of promoter libraries, developmental and evolutionary lineage tracking, and various other exploratory assays. However, the need for library transgenesis has effectively restricted these approaches to single-cell models. Here we present Transgenic Arrays Resulting in Diversity of Integrated Sequences (TARDIS), a simple yet powerful approach to large-scale transgenesis that overcomes typical limitations encountered in multicellular systems. TARDIS splits the transgenesis process into a two-step process: creation of individuals carrying experimentally introduced sequence libraries, followed by inducible extraction and integration of individual sequences/library components from the larger library cassette into engineered genomic sites. Thus, transformation of a single individual, followed by lineage expansion and functional transgenesis, gives rise to thousands of genetically unique transgenic individuals. We demonstrate the power of this system using engineered, split selectable TARDIS sites in Caenorhabditis elegans to generate (1) a large set of individually barcoded lineages and (2) transcriptional reporter lines from pre-defined promoter libraries. We find that this approach increases transformation yields up to approximately 1000-fold over current single-step methods. While we demonstrate the utility of TARDIS using C. elegans, in principle the process is adaptable to any system where experimentally generated genomic loci landing pads and diverse, heritable DNA elements can be generated.

genetics↗

The coupling between healthspan and lifespan in Caenorhabditis depends on complex interactions between compound intervention and genetic background

Aging is characterized by declining health that results in decreased neuromuscular function and cellular resilience. The relationship between lifespan and health, and the influence of genetic background on that relationship, has important implications in the development of anti-aging interventions. Here we combined survival under thermal and oxidative stress with swimming performance, to evaluate health effects across a nematode genetic diversity panel for three compounds previously studied in the Caenorhabditis Intervention Testing Program - NP1, propyl gallate, and resveratrol. We show that oxidative stress resistance and thermotolerance vary with compound intervention, genetic background, and age. The effects of tested compounds on swimming locomotion, in contrast, are largely species-specific. Additionally, thermotolerance, but not oxidative stress or swimming ability, correlates with lifespan. Our results demonstrate the importance of assessing health and lifespan across genetic backgrounds in the effort to identify reproducible aging interventions.

physiology↗

Alignment of genetic differentiation across trophic levels in a fig community

Ecological interactions can generate close associations among species, which can in turn generate a high degree of overlap in their spatial distributions. Co-occurrence is likely to be particularly intense when species exhibit obligate comigration, in which they not only overlap in spatial distributions but also travel together from patch to patch. In theory, this pattern of ecological co-occurrence should leave a distinct signature in the pattern of genetic differentiation within and among species. Perhaps the most famous mutual co-isolation partners are fig trees and their co-evolved wasp pollinators. Here, we add another tropic level to this system by examining patterns of genomic diversity in the nematode Caenorhabditis inopinata, a close relative of the C. elegans model system that thrives in figs and obligately disperses on fig wasps. We performed RADseq on individual worms isolated from the field across three Okinawan island populations. The male/female C. inopinata is about five times more diverse than the hermaphroditic C. elegans, and polymorphism is enriched on chromosome arms relative to chromosome centers. FST is low among island population pairs, and clear population structure could not be easily detected among figs, trees, and islands, suggesting frequent migration of wasps between islands. Moreover, inbreeding coefficients are elevated in C. inopinata, consistent with field observations suggesting small C. inopinata founding populations in individual figs. These genetic patterns in C. inopinata overlap with those previously reported in its specific fig wasp vector and are consistent with C. inopinata population dynamics being driven by wasp dispersal. Thus, interspecific interactions can align patterns of genetic diversity across species separated by hundreds of millions of years of evolutionary divergence. HighlightsO_LIThe fig-dwelling female/male nematode Caenorhabditis inopinata is five times more diverse than its closest relative, the self-fertilizing nematode C. elegans. C_LIO_LIC. inopinata migrates frequently among three Okinawan islands despite high levels of inbreeding within individual figs. C_LIO_LIC. inopinata has patterns of genetic diversity that mirror its fig wasp vector. C_LIO_LIEcological specialization aligns patterns of genetic differentiation in closely interacting species. C_LI

evolutionary biology↗

Antioxidants green tea extract and nordihydroguaiaretic acid confer species and strain specific lifespan and health effects in Caenorhabditis nematodes

The Caenorhabditis Intervention Testing Program (CITP) is an NIH-funded research consortium of investigators who conduct analyses at three independent sites to identify chemical interventions that reproducibly promote health and lifespan in a robust manner. The founding principle of the CITP is that compounds with positive effects across a genetically diverse panel of Caenorhabditis species and strains are likely engaging conserved biochemical pathways to exert their effects. As such, interventions that are broadly efficacious might be considered prominent compounds for translation for pre-clinical research and human clinical applications. Here, we report results generated using a recently streamlined pipeline approach for the evaluation of the effects of chemical compounds on lifespan and health. We studied five compounds previously shown to extend C. elegans lifespan or thought to promote mammalian health: 17-estradiol, acarbose, green tea extract, nordihydroguaiaretic acid, and rapamycin. We found that green tea extract and nordihydroguaiaretic acid extend Caenorhabditis lifespan in a species-specific manner. Additionally, these two antioxidants conferred assay-specific effects in some studies--for example, decreasing survival for certain genetic backgrounds in manual survival assays in contrast with extended lifespan as assayed using automated C. elegans Lifespan Machines. We also observed that GTE and NDGA impact on older adult mobility capacity is dependent on genetic background, and that GTE reduces oxidative stress resistance in some Caenorhabditis strains. Overall, our analysis of the five compounds supports the general idea that genetic background and assay type can influence lifespan and health effects of compounds, and underscores that lifespan and health can be uncoupled by chemical interventions.

physiology↗

High-throughput imaging of Caenorhabditis elegans aging using collective activity monitoring

The genetic manipulability and short lifespan of C. elegans make it an important model for aging research. Widely applied methods for measurements of worm aging based on manual observation are labor intensive and low-throughput. Here, we describe the Worm Collective Activity Monitoring Platform (WormCamp), a system for assaying aging in C. elegans by monitoring activity of populations of worms in standard 24-well plates. We show that metrics based on the rate of decline in collective activity can be used to estimate the average lifespan and locomotor healthspan in the population. Using the WormCamp, we assay a panel of highly divergent natural isolates of C. elegans and show that both lifespan and locomotor healthspan display substantial heritability. To facilitate analysis of large numbers of worms, we developed a robotic imaging system capable of simultaneous automated monitoring of activity, lifespan, and locomotor healthspan in up to 2,304 populations containing a total of ~90,000 animals. We applied the automated system to conduct a large-scale RNA interference screen for genes that affect lifespan and locomotor healthspan. The WormCamp system is complementary to other current automated methods for assessing C. elegans aging and is well suited for efficiently screening large numbers of conditions.

bioengineering↗

Post-Insemination Selection Dominates Pre-Insemination Selection in Driving Rapid Evolution of Male Competitive Ability

Sexual reproduction is a complex process that contributes to differences between the sexes and divergence between species. From a males perspective, sexual selection can optimize reproductive success by acting on the variance in mating success (pre-insemination selection) as well as the variance in fertilization success (post-insemination selection). The balance between pre- and post-insemination selection has not yet been investigated using a strong hypothesis-testing framework that directly quantifies the effects of post-insemination selection on the evolution of reproductive success. Here we use experimental evolution of a uniquely engineered genetic system that allows sperm production to be turned off and on in obligate male-female populations of Caenorhabditis elegans. We show that enhanced post-insemination competition increases the efficacy of selection and surpasses pre-insemination sexual selection in driving a polygenic response in male reproductive success. We find that after 30 generations post-insemination selection increased male reproductive success by an average of 5- to 7-fold. Contrary to expectation, enhanced pre-insemination competition hindered selection and slowed the rate of evolution. Furthermore, we found that post-insemination selection resulted in a strong polygenic response at the whole-genome level. Our results demonstrate that post-insemination sexual selection plays a critical role in the rapid optimization of male reproductive fitness. Therefore, explicit consideration should be given to post-insemination dynamics when considering the population effects of sexual selection.

evolutionary biology↗