From whole bodies to single cells: A guide to transcriptomic approaches for ecology and evolutionary biology.

RNA sequencing (RNAseq) methodology has experienced a burst of technological developments in the last decade, which has opened up opportunities for studying the mechanisms of adaptation to environmental factors at both the organismal and cellular level. Selecting the most suitable experimental approach for specific research questions and model systems can, however, be a challenge and researchers in ecology and evolution are commonly faced with the choice of whether to study gene expression variation in whole bodies, specific tissues, and/or single cells. A wide range of sometimes polarised opinions exists over which approach is best. Here, we highlight the advantages and disadvantages of each of these approaches to provide a guide to help researchers make informed decisions and maximise the power of their study. Using illustrative examples of various ecological and evolutionary research questions, we guide the readers through the different RNAseq approaches and help them identify the most suitable design for their own projects.

Pleiotropic fitness effects across sexes and ages in the Drosophila genome and transcriptome.

Selection varies between categories of individuals, with far-reaching ramifications: Sex-specific selection can impede or accelerate adaptation, and differences in selection between young and old individuals are ultimately responsible for senescence. Here, we measure early- and late-life fitness in adults of both sexes from the Drosophila genetic reference panel and perform quantitative genetic and transcriptomic analyses. Fitness was heritable, showed positive pleiotropy across sexes and age classes, and appeared to be influenced by very large numbers of loci with small effects plus a smaller number with moderate effects. Most loci affected male and female fitness in the same direction; relatively few candidate sexually antagonistic loci were found, though these were enriched on the X chromosome as predicted by theory. The expression level of many genes showed an opposite correlation with fitness in males and females, consistent with unresolved sexual conflict over transcription. The load of deleterious mutations correlated negatively with fitness across genotypes, and we found some evidence for the mutation accumulation (but not the antagonistic pleiotropy) theory of aging.

Polygenic signals of sex differences in selection in humans from the UK Biobank.

Sex differences in the fitness effects of genetic variants can influence the rate of adaptation and the maintenance of genetic variation. For example, "sexually antagonistic" (SA) variants, which are beneficial for one sex and harmful for the other, can both constrain adaptation and increase genetic variability for fitness components such as survival, fertility, and disease susceptibility. However, detecting variants with sex-differential fitness effects is difficult, requiring genome sequences and fitness measurements from large numbers of individuals. Here, we develop new theory for studying sex-differential selection across a complete life cycle and test our models with genotypic and reproductive success data from approximately 250,000 UK Biobank individuals. We uncover polygenic signals of sex-differential selection affecting survival, reproductive success, and overall fitness, with signals of sex-differential reproductive selection reflecting a combination of SA polymorphisms and sexually concordant polymorphisms in which the strength of selection differs between the sexes. Moreover, these signals hold up to rigorous controls that minimise the contributions of potential confounders, including sequence mapping errors, population structure, and ascertainment bias. Functional analyses reveal that sex-differentiated sites are enriched in phenotype-altering genomic regions, including coding regions and loci affecting a range of quantitative traits. Population genetic analyses show that sex-differentiated sites exhibit evolutionary histories dominated by genetic drift and/or transient balancing selection, but not long-term balancing selection, which is consistent with theoretical predictions of effectively weak SA balancing selection in historically small populations. Overall, our results are consistent with polygenic sex-differential-including SA-selection in humans. Evidence for sex-differential selection is particularly strong for variants affecting reproductive success, in which the potential contributions of nonrandom sampling to signals of sex differentiation can be excluded.

Detection of community-wide impacts of bottom trawl fishing on deep-sea assemblages using environmental DNA metabarcoding.

Although considerable research progress on the effects of anthropogenic disturbance in the deep sea has been made in recent years, our understanding of these impacts at community level remains limited. Here, we studied deep-sea assemblages of Sicily (Mediterranean Sea) subject to different intensities of benthic trawling using environmental DNA (eDNA) metabarcoding and taxonomic identification of meiofauna communities. Firstly, eDNA metabarcoding data did not detect trawling impacts using alpha diversity whereas meiofauna data detected a significant effect of trawling. Secondly, both eDNA and meiofauna data detected significantly different communities across distinct levels of trawling intensity when we examined beta diversity. Taxonomic assignment of the eDNA data revealed that Bryozoa was present only at untrawled sites, highlighting their vulnerability to trawling. Our results provide evidence for community-wide impacts of trawling, with different trawling intensities leading to distinct deep-sea communities. Finally, we highlight the need for further studies to unravel understudied deep-sea biodiversity.

Experimental sexual selection affects the evolution of physiological and life-history traits.

Sexual selection and sexual conflict are expected to affect all aspects of the phenotype, not only traits that are directly involved in reproduction. Here, we show coordinated evolution of multiple physiological and life-history traits in response to long-term experimental manipulation of the mating system in populations of Drosophila pseudoobscura. Development time was extended under polyandry relative to monogamy in both sexes, potentially due to higher investment in traits linked to sexual selection and sexual conflict. Individuals (especially males) evolving under polyandry had higher metabolic rates and locomotor activity than those evolving under monogamy. Polyandry individuals also invested more in metabolites associated with increased endurance capacity and efficient energy metabolism and regulation, namely lipids and glycogen. Finally, polyandry males were less desiccation- and starvation resistant than monogamy males, suggesting trade-offs between resistance and sexually selected traits. Our results provide experimental evidence that mating systems can impose selection that influences the evolution of non-sexual phenotypes such as development, activity, metabolism and nutrient homeostasis.

Managing human-mediated range shifts: understanding spatial, temporal and genetic variation in marine non-native species.

The use of molecular tools to manage natural resources is increasingly common. However, DNA-based methods are seldom used to understand the spatial and temporal dynamics of species' range shifts. This is important when managing range shifting species such as non-native species (NNS), which can have negative impacts on biotic communities. Here, we investigated the ascidian NNS Ciona robusta, Clavelina lepadiformis, Microcosmus squamiger and Styela plicata using a combined methodological approach. We first conducted non-molecular biodiversity surveys for these NNS along the South African coastline, and compared the results with historical surveys. We detected no consistent change in range size across species, with some displaying range stability and others showing range shifts. We then sequenced a section of cytochrome c oxidase subunit I (COI) from tissue samples and found genetic differences along the coastline but no change over recent times. Finally, we found that environmental DNA metabarcoding data showed broad congruence with both the biodiversity survey and the COI datasets, but failed to capture the complete incidence of all NNS. Overall, we demonstrated how a combined methodological approach can effectively detect spatial and temporal variation in genetic composition and range size, which is key for managing both thriving NNS and threatened species. This article is part of the theme issue 'Species' ranges in the face of changing environments (part I)'.

A comment on The adaptive value of gluttony: predators mediate the life history trade-offs of satiation threshold by Pruitt & Krauel (2010).

Inspection of the data that accompany Pruitt and Krauel's study of individual variation in satiation threshold and a comparison of these data with the Materials and Methods and Results sections of the paper have revealed a number of issues that cast doubts on the reliability of the data and any results based on these data. In particular, we show that, following our analyses, the data are unlikely to have been obtained using the study design outlined in the publication and that statistical analyses of these data provide results that differ in important ways from those reported. These findings illustrate the importance of making raw data and analysis code available for the rigour and reproducibility of the scientific literature.

Sexual selection can partly explain low frequencies of Segregation Distorter alleles.

The Segregation Distorter (SD) allele found in Drosophila melanogaster distorts Mendelian inheritance in heterozygous males by causing developmental failure of non-SD spermatids, such that greater than 90% of the surviving sperm carry SD. This within-individual advantage should cause SD to fix, and yet SD is typically rare in wild populations. Here, we explore whether this paradox can be resolved by sexual selection, by testing if males carrying three different variants of SD suffer reduced pre- or post-copulatory reproductive success. We find that males carrying the SD allele are just as successful at securing matings as control males, but that one SD variant (SD-5) reduces sperm competitive ability and increases the likelihood of female remating. We then used these results to inform a theoretical model; we found that sexual selection could limit SD to natural frequencies when sperm competitive ability and female remating rate equalled the values observed for SD-5. However, sexual selection was unable to explain natural frequencies of the SD allele when the model was parameterized with the values found for two other SD variants, indicating that sexual selection alone is unlikely to explain the rarity of SD.

Environmental DNA sampling protocols for the surveillance of marine non-indigenous species in Irish coastal waters.

Understanding the spread and distribution of Non-Indigenous Species (NIS) is key when implementing legislation to maintain good ecosystem health. Environmental DNA (eDNA) has shown great potential to detect aquatic organisms in a rapid and cost-effective way, however their applicability to new environments must be validated prior to their implementation. Here, we tested different field sampling methods in combination with eDNA metabarcoding to develop a tool to detect NIS. Large and small volumes of seawater were filtered, in addition to the collection of sediment and horizontal tow net samples at 12 locations across four distinct geographic areas in Ireland. The biggest dissimilarity in the species recovered was found between sediment and town net samples. Tow nets showed to be the most efficient. A total of 357 taxa were identified, including 16 NIS. Fine mesh tow nets were identified as the most cost-efficient for large-scale monitoring and surveillance of NIS.

Trade-offs between reducing complex terminology and producing accurate interpretations from environmental DNA: Comment on "Environmental DNA: What's behind the term?" by Pawlowski et al., (2020).

In a recent paper, "Environmental DNA: What's behind the term? Clarifying the terminology and recommendations for its future use in biomonitoring," Pawlowski et al. argue that the term eDNA should be used to refer to the pool of DNA isolated from environmental samples, as opposed to only extra-organismal DNA from macro-organisms. We agree with this view. However, we are concerned that their proposed two-level terminology specifying sampling environment and targeted taxa is overly simplistic and might hinder rather than improve clear communication about environmental DNA and its use in biomonitoring. This terminology is based on categories that are often difficult to assign and uninformative, and it overlooks a fundamental distinction within eDNA: the type of DNA (organismal or extra-organismal) from which ecological interpretations are derived.

Animals, protists and bacteria share marine biogeographic patterns.

Over millennia, ecological and evolutionary mechanisms have shaped macroecological patterns across the tree of life. Research describing these patterns at both regional and global scales has traditionally focused on the study of metazoan species. Consequently, there is a limited understanding of cross-phylum biogeographic structuring and an escalating need to understand the macroecology of both microscopic and macroscopic organisms. Here we used environmental DNA (eDNA) metabarcoding to explore the biodiversity of marine metazoans, protists and bacteria along an extensive and highly heterogeneous coastline. Our results showed remarkably consistent biogeographic structure across the kingdoms of life despite billions of years of evolution. Analyses investigating the drivers of these patterns for each taxonomic kingdom found that environmental conditions (such as temperature) and, to a lesser extent, anthropogenic stressors (such as fishing pressure and pollution) explained some of the observed variation. Additionally, metazoans displayed biogeographic patterns that suggested regional biotic homogenization. Against the backdrop of global pervasive anthropogenic environmental change, our work highlights the importance of considering multiple domains of life to understand the maintenance and drivers of biodiversity patterns across broad taxonomic, ecological and geographical scales.

Male-biased sexual selection, but not sexual dichromatism, predicts speciation in birds.

Sexual selection is thought to shape phylogenetic diversity by affecting speciation or extinction rates. However, the net effect of sexual selection on diversification is hard to predict because many of the hypothesized effects on speciation or extinction have opposing signs and uncertain magnitudes. Theoretical work also suggests that the net effect of sexual selection on diversification should depend strongly on ecological factors, though this prediction has seldom been tested. Here, we test whether variation in sexual selection can predict speciation and extinction rates across passerine birds (up to 5812 species, covering most genera) and whether this relationship is mediated by environmental factors. Male-biased sexual selection, and specifically sexual size dimorphism, predicted two of the three measures of speciation rates that we examined. The link we observed between sexual selection and speciation was independent of environmental variability, though species with smaller ranges had higher speciation rates. There was no association between any proxies of sexual selection and extinction rate. Our findings support the view that male-biased sexual selection, as measured by frequent predictors of male-male competition, has shaped diversification in the largest radiation of birds.

Resistance to natural and synthetic gene drive systems.

Scientists are rapidly developing synthetic gene drive elements intended for release into natural populations. These are intended to control or eradicate disease vectors and pests, or to spread useful traits through wild populations for disease control or conservation purposes. However, a crucial problem for gene drives is the evolution of resistance against them, preventing their spread. Understanding the mechanisms by which populations might evolve resistance is essential for engineering effective gene drive systems. This review summarizes our current knowledge of drive resistance in both natural and synthetic gene drives. We explore how insights from naturally occurring and synthetic drive systems can be integrated to improve the design of gene drives, better predict the outcome of releases and understand genomic conflict in general.

Sibling rivalry versus mother's curse: can kin competition facilitate a response to selection on male mitochondria?

Assuming that fathers never transmit mitochondrial DNA (mtDNA) to their offspring, mitochondrial mutations that affect male fitness are invisible to direct selection on males, leading to an accumulation of male-harming alleles in the mitochondrial genome (mother's curse). However, male phenotypes encoded by mtDNA can still undergo adaptation via kin selection provided that males interact with females carrying related mtDNA, such as their sisters. Here, using experiments with Drosophila melanogaster carrying standardized nuclear DNA but distinct mitochondrial DNA, we test whether the mitochondrial haplotype carried by interacting pairs of larvae affects survival to adulthood, as well as the fitness of the adults. Although mtDNA had no detectable direct or indirect genetic effect on larva-to-adult survival, the fitness of male and female adults was significantly affected by their own mtDNA and the mtDNA carried by their social partner in the larval stage. Thus, mtDNA mutations that alter the effect of male larvae on nearby female larvae (which often carry the same mutation, due to kinship) could theoretically respond to kin selection. We discuss the implications of our findings for the evolution of mitochondria and other maternally inherited endosymbionts.

Publisher Correction: Detection of introduced and resident marine species using environmental DNA metabarcoding of sediment and water.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Fitness consequences of the selfish supergene Segregation Distorter.

Segregation distorters are selfish genetic elements that subvert Mendelian inheritance, often by destroying gametes that do not carry the distorter. Simple theoretical models predict that distorter alleles will either spread to fixation or stabilize at some high intermediate frequency. However, many distorters have substantially lower allele frequencies than predicted by simple models, suggesting that key sources of selection remain to be discovered. Here, we measured the fitness of Drosophila melanogaster adults and juveniles carrying zero, one or two copies of three different variants of the naturally occurring supergene Segregation Distorter (SD), in order to investigate why SD alleles remain relatively rare within populations despite being preferentially inherited. First, we show that the three SD variants differ in the severity and dominance of the fitness costs they impose on individuals carrying them. Second, SD-carrying parents produced less fit offspring in some crosses, independent of offspring genotype, indicating that SD alleles can have nongenetic, transgenerational costs in addition to their direct costs. Third, we found that SD carriers sometimes produce a biased offspring sex ratio, perhaps due to off-target effects of SD on the sex chromosomes. Finally, we used a theoretical model to investigate how sex ratio and transgenerational effects alter the population genetics of distorter alleles; accounting for these additional costs helps to explain why real-world segregation distorter alleles are rarer than predicted.

Mother's curse and indirect genetic effects: Do males matter to mitochondrial genome evolution?

Maternal inheritance of mitochondrial DNA (mtDNA) was originally thought to prevent any response to selection on male phenotypic variation attributable to mtDNA, resulting in a male-biased mtDNA mutation load ("mother's curse"). However, the theory underpinning this claim implicitly assumes that a male's mtDNA has no effect on the fitness of females he comes into contact with. If such "mitochondrially encoded indirect genetics effects" (mtIGEs) do in fact exist, and there is relatedness between the mitochondrial genomes of interacting males and females, male mtDNA-encoded traits can undergo adaptation after all. We tested this possibility using strains of Drosophila melanogaster that differ in their mtDNA. Our experiments indicate that female fitness is influenced by the mtDNA carried by males that the females encounter, which could plausibly allow the mitochondrial genome to evolve via kin selection. We argue that mtIGEs are probably common, and that this might ameliorate or exacerbate mother's curse.

Demonstration of the Use of Environmental DNA for the Non-Invasive Genotyping of a Bivalve Mollusk, the European Flat Oyster (Ostrea edulis).

Accurate SNP (single nucleotide polymorphism) genotype information is critical for a wide range of selective breeding applications in aquaculture, including parentage assignment, marker-assisted, and genomic selection. However, the sampling of tissue for genetic analysis can be invasive for juvenile animals or taxa where sampling tissue is difficult or may cause mortality (e.g. bivalve mollusks). Here, we demonstrate a novel, non-invasive technique for sampling DNA based on the collection of environmental DNA using European Flat Oysters (Ostrea edulis) as an example. The live animals are placed in individual containers until sufficient genetic material is released into the seawater which is then recovered by filtration. We compared the results of tissue and eDNA derived SNP genotype calls using a PCR based genotyping platform. We found that 100% accurate genotype calls from eDNA are possible, but depend on appropriate filtration and the dilution of the sample throughout the workflow. We also developed an additional low-cost DNA extraction technique which provided >99% correct SNP genotype calls in comparison to tissue. It was concluded that eDNA sampling can be used in hatchery and selective breeding programs applicable to any aquatic organism for which direct tissue sampling may result in animal welfare concerns or mortality.

An X-linked meiotic drive allele has strong, recessive fitness costs in female Drosophila pseudoobscura.

Selfish 'meiotic drive' alleles are transmitted to more than 50% of offspring, allowing them to rapidly invade populations even if they reduce the fitness of individuals carrying them. Theory predicts that drivers should either fix or go extinct, yet some drivers defy these predictions by persisting at low, stable frequencies for decades. One possible explanation for this discrepancy is that drivers are especially costly when homozygous, although empirical tests of this idea are rare and equivocal. Here, we measure the fitness of female Drosophila pseudoobscura carrying zero, one or two copies of the X-linked driver sex ratio (SR). SR had strong negative effects on female offspring production and the probability of reproductive failure, and these effects were largely similar across four genetic backgrounds. SR was especially costly when homozygous. We used our fitness measurements to parametrize a population genetic model, and found that the female fitness costs observed here can explain the puzzlingly low allele frequency of SR in nature. We also use the model to show how spatial variation in female mating behaviour, fitness costs of SR and the reduced siring success of SR males can jointly explain the north-south cline in SR frequencies across North America.