Reproductive system, temperature, and genetic background effects in experimentally evolving populations of Caenorhabditis elegans.

Experimental evolution (EE) is a powerful research framework for gaining insights into many biological questions, including the evolution of reproductive systems. We designed a long-term and highly replicated EE project using the nematode C. elegans, with the main aim of investigating the impact of reproductive system on adaptation and diversification under environmental challenge. From the laboratory-adapted strain N2, we derived isogenic lines and introgressed the fog-2(q71) mutation, which changes the reproductive system from nearly exclusive selfing to obligatory outcrossing, independently into 3 of them. This way, we obtained 3 pairs of isogenic ancestral populations differing in reproductive system; from these, we derived replicate EE populations and let them evolve in either novel (increased temperature) or control conditions for over 100 generations. Subsequently, fitness of both EE and ancestral populations was assayed under the increased temperature conditions. Importantly, each population was assayed in 2-4 independent blocks, allowing us to gain insight into the reproducibility of fitness scores. We expected to find upward fitness divergence, compared to ancestors, in populations which had evolved in this treatment, particularly in the outcrossing ones due to the benefits of genetic shuffling. However, our data did not support these predictions. The first major finding was very strong effect of replicate block on populations' fitness scores. This indicates that despite standardization procedures, some important environmental effects were varying among blocks, and possibly compounded by epigenetic inheritance. Our second key finding was that patterns of EE populations' divergence from ancestors differed among the ancestral isolines, suggesting that research conclusions derived for any particular genetic background should never be generalized without sampling a wider set of backgrounds. Overall, our results support the calls to pay more attention to biological variability when designing studies and interpreting their results, and to avoid over-generalizations of outcomes obtained for specific genetic and/or environmental conditions.

Evolution of fertilization ability in obligatorily outcrossing populations of Caenorhabditis elegans.

In species reproducing by selfing, the traits connected with outcrossing typically undergo degeneration, a phenomenon called selfing syndrome. In Caenorhabditis elegans nematodes, selfing syndrome affects many traits involved in mating, rendering cross-fertilization highly inefficient. In this study, we investigated the evolution of cross-fertilization efficiency in populations genetically modified to reproduce by obligatory outcrossing. Following the genetic modification, replicate obligatorily outcrossing were maintained for over 100 generations, at either optimal (20 °C) or elevated (24 °C) temperatures, as a part of a broader experimental evolution program. Subsequently, fertilization rates were assayed in the evolving populations, as well as their ancestors who had the obligatory outcrossing introduced but did not go through experimental evolution. Fertilization effectivity was measured by tracking the fractions of fertilized females in age-synchronized populations, through 8 h since reaching adulthood. In order to check the robustness of our measurements, each evolving population was assayed in two or three independent replicate blocks. Indeed, we found high levels of among-block variability in the fertilization trajectories, and in the estimates of divergence between evolving populations and their ancestors. We also identified five populations which appear to have evolved increased fertilization efficiency, relative to their ancestors. However, due to the abovementioned high variability, this set of populations should be treated as candidate, with further replications needed to either confirm or disprove their divergence from ancestors. Furthermore, we also discuss additional observations we have made concerning fertilization trajectories.

Does operational sex ratio influence relative strength of purging selection in males versus females?

According to theory, sexual selection in males may efficiently purge mutation load of sexual populations, reducing or fully compensating 'the cost of males'. For this to occur, mutations not only need to be deleterious to both sexes, they also must affect males more than females. A frequently overlooked problem is that relative strength of selection on males versus females may vary between environments, with social conditions being particularly likely to affect selection in males and females differently. Here, we induced mutations in red flour beetles (Tribolium castaneum) and tested their effect in both sexes under three different operational sex ratios (1:2, 1:1 and 2:1). Induced mutations decreased fitness of both males and females, but their effect was not stronger in males. Surprisingly, operational sex ratio did not affect selection against deleterious mutations nor its relative strength in the sexes. Thus, our results show no support for the role of sexual selection in the evolutionary maintenance of sex.

Competitive fitness analysis using Convolutional Neural Network.

We developed a procedure for estimating competitive fitness by using Caenorhabditis elegans as a model organism and a Convolutional Neural Network (CNN) as a tool. Competitive fitness is usually the most informative fitness measure, and competitive fitness assays often rely on green fluorescent protein (GFP) marker strains. CNNs are a class of deep learning neural networks, which are well suited for image analysis and object classification. Our model analyses involved image classification of nematodes as wild-type vs. GFP-expressing, and counted both categories. The performance was analyzed with (i) precision and recall parameters, and (ii) comparison of the wild-type frequency calculated from the model against that obtained by visual scoring of the same images. The average precision and recall varied from 0.79 to 0.87 and from 0.84 to 0.92, respectively, depending on worm density in the images. Compared with manual counting, the model decreased counting time at least 20-fold while preventing human errors. Given the rapid development in the field of CNN, the model, which is fully available on GitHub, can be further optimized and adapted for other image-based uses.

Sexual dimorphism in immunity across animals: a meta-analysis.

In animals, sex differences in immunity are proposed to shape variation in infection prevalence and intensity among individuals in a population, with females typically expected to exhibit superior immunity due to life-history trade-offs. We performed a systematic meta-analysis to investigate the magnitude and direction of sex differences in immunity and to identify factors that shape sex-biased immunocompetence. In addition to considering taxonomic and methodological effects as moderators, we assessed age-related effects, which are predicted to occur if sex differences in immunity are due to sex-specific resource allocation trade-offs with reproduction. In a meta-analysis of 584 effects from 124 studies, we found that females exhibit a significantly stronger immune response than do males, but the effect size is relatively small, and became non-significant after controlling for phylogeny. Female-biased immunity was more pronounced in adult than immature animals. More recently published studies did not report significantly smaller effect sizes. Among taxonomic and methodological subsets of the data, some of the largest effect sizes were in insects, further supporting previous suggestions that testosterone is not the only potential driver of sex differences in immunity. Our findings challenge the notion of pervasive biases towards female-biased immunity and the role of testosterone in driving these differences.

Fitness Effects of Thermal Stress Differ Between Outcrossing and Selfing Populations in Caenorhabditis elegans.

The maintenance of males and outcrossing is widespread, despite considerable costs of males. By enabling recombination between distinct genotypes, outcrossing may be advantageous during adaptation to novel environments and if so, it should be selected for under environmental challenge. However, a given environmental change may influence fitness of male, female, and hermaphrodite or asexual individuals differently, and hence the relationship between reproductive system and dynamics of adaptation to novel conditions may not be driven solely by the level of outcrossing and recombination. This has important implications for studies investigating the evolution of reproductive modes in the context of environmental changes, and for the extent to which their findings can be generalized. Here, we use Caenorhabditis elegans-a free-living nematode species in which hermaphrodites (capable of selfing but not cross-fertilizing each other) coexist with males (capable of fertilizing hermaphrodites)-to investigate the response of wild type as well as obligatorily outcrossing and obligatorily selfing lines to stressfully increased ambient temperature. We found that thermal stress affects fitness of outcrossers much more drastically than that of selfers. This shows that apart from the potential for recombination, the selective pressures imposed by the same environmental change can differ between populations expressing different reproductive systems and affect their adaptive potential.

Do males pay for sex? Sex-specific selection coefficients suggest not.

Selection acting on males can reduce mutation load of sexual relative to asexual populations, thus mitigating the twofold cost of sex, provided that it seeks and destroys the same mutations as selection acting on females, but with higher efficiency. This could happen due to sexual selection-a potent evolutionary force that in most systems predominantly affects males. We used replicate populations of red flour beetles (Tribolium castaneum) to study sex-specific selection against deleterious mutations introduced with ionizing radiation. We found no evidence for selection being stronger in males than in females; in fact, we observed a nonsignificant trend in the opposite direction. This suggests that selection on males does not reduce mutation load below the level expected under the (hypothetical) scenario of asexual reproduction. Additionally, we employed a novel approach, based on a simple model, to quantify the relative contributions of sexual and offspring viability selection to the overall selection observed in males. We found them to be similar in magnitude; however, only the offspring viability component was statistically significant. In summary, we found no support for the hypothesis that selection on males in general, and sexual selection in particular, contributes to the evolutionary maintenance of sex.

Genetic variation in male attractiveness: It is time to see the forest for the trees.

Female choice based on multiple male traits, rather than on any single one, has been reported in many species and may well be a rule rather than an exception. However, the implications this has for selection acting on choosiness itself remain underappreciated. We argue that this constitutes one of the important impediments to our understanding of the evolution of mate choice. We discuss this issue primarily in the context of the Fisherian model of sexual selection. We review theory and empirical data, showing how the crucial parameter of the model-genetic variation in male attractiveness-can be estimated when attractiveness is a function of multiple traits. Based on the reviewed theory, we show how relying on individual male traits, instead of overall attractiveness, can produce biased estimates of Fisherian benefits of female choice. This bias can be substantial, especially when many traits contribute to male attractiveness. We discuss a number of methodological issues that, we hope, will stimulate future studies and help resolving the long-standing mystery of mate choice.

No evidence for reproductive isolation through sexual conflict in the bulb mite Rhizoglyphus robini.

Sexual conflict leading to sexual antagonistic coevolution has been hypothesized to drive reproductive isolation in allopatric populations and hence lead to speciation. However, the generality of this speciation mechanism is under debate. We used experimental evolution in the bulb mite Rhizoglyphusrobini to investigate whether sexual conflict promotes reproductive isolation measured comprehensively to include all possible pre- and post-zygotic mechanisms. We established replicate populations in which we either enforced monogamy, and hence removed sexual conflict by making male and female evolutionary interests congruent, or allowed promiscuity. After 35 and 45 generations of experimental evolution, we found no evidence of reproductive isolation between the populations in any of the mating systems. Our results indicate that sexual conflict does not necessarily drive fast reproductive isolation and it may not be a ubiquitous mechanism leading to speciation.

Mating system affects population performance and extinction risk under environmental challenge.

Failure of organisms to adapt to sudden environmental changes may lead to extinction. The type of mating system, by affecting fertility and the strength of sexual selection, may have a major impact on a population's chances to adapt and survive. Here, we use experimental evolution in bulb mites (Rhizoglyphus robini) to examine the effects of the mating system on population performance under environmental change. We demonstrate that populations in which monogamy was enforced suffered a dramatic fitness decline when evolving at an increased temperature, whereas the negative effects of change in a thermal environment were alleviated in polygamous populations. Strikingly, within 17 generations, all monogamous populations experiencing higher temperature went extinct, whereas all polygamous populations survived. Our results show that the mating system may have dramatic effects on the risk of extinction under environmental change.

Meta-analysis suggests choosy females get sexy sons more than "good genes".

Female preferences for specific male phenotypes have been documented across a wide range of animal taxa, including numerous species where males contribute only gametes to offspring production. Yet, selective pressures maintaining such preferences are among the major unknowns of evolutionary biology. Theoretical studies suggest that preferences can evolve if they confer genetic benefits in terms of increased attractiveness of sons ("Fisherian" models) or overall fitness of offspring ("good genes" models). These two types of models predict, respectively, that male attractiveness is heritable and genetically correlated with fitness. In this meta-analysis, we draw general conclusions from over two decades worth of empirical studies testing these predictions (90 studies on 55 species in total). We found evidence for heritability of male attractiveness. However, attractiveness showed no association with traits directly associated with fitness (life-history traits). Interestingly, it did show a positive correlation with physiological traits, which include immunocompetence and condition. In conclusion, our results support "Fisherian" models of preference evolution, while providing equivocal evidence for "good genes." We pinpoint research directions that should stimulate progress in our understanding of the evolution of female choice.