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Key Points The hypothesis that differences in gene regulation have an important role in speciation and adaptation is more than 40 years old. RNA sequencing (RNA-seq) allows measurement and comparison of gene expression levels across practically any combination of species at an unprecedented resolution. Comparative studies of gene expression levels in all species studied to date provide compelling evidence that most gene regulatory patterns evolve under evolutionary constraint. It is more difficult to infer the action of positive (directional) selection on gene regulation than the action of stabilizing selection, especially in non-model species such as humans and non-human apes, where environmental and genetic effects might be confounded. Inter-species differences in epigenetic markers can probably explain a substantial fraction of gene expression differences between species. Because a broad range of experimental manipulations are possible in model organisms, studies that focus on model species can move beyond simple comparisons of gene expression and offer deep insights into the causal relationship between regulatory changes and phenotypic evolution. Functional studies in model systems can often shed light on the adaptive phenotypes that were affected by regulatory changes between humans and other primates. Some phenotypes, however (for example, the development of language), are inherently difficult to study using model species. It might be possible to use induced pluripotent stem cells derived differentiated cells from humans and non-human primates to test functionally for the outcomes of inter-species differences in gene regulation. Gene expression levels can now be compared among species at greater resolution. Focusing on work in primates, the authors discuss the evolution of gene expression, ways of exploring mechanisms that underlie expression changes and complementary work in model organisms on the functional effects of expression changes. The hypothesis that differences in gene regulation have an important role in speciation and adaptation is more than 40 years old. With the advent of new sequencing technologies, we are able to characterize and study gene expression levels and associated regulatory mechanisms in a large number of individuals and species at an unprecedented resolution and scale. We have thus gained new insights into the evolutionary pressures that shape gene expression levels and have developed an appreciation for the relative importance of evolutionary changes in different regulatory genetic and epigenetic mechanisms. The current challenge is to link gene regulatory changes to adaptive evolution of complex phenotypes. Here we mainly focus on comparative studies in primates and how they are complemented by studies in model organisms.

Pheromones are secreted molecules that mediate animal communications. These olfactory signals can have substantial effects on physiology and likely play important roles in organismal survival in natural habitats. Here we show that a blend of two ascaroside pheromones produced by C. elegans males primes the female reproductive system in part by improving sperm guidance toward oocytes. Worms have different physiological responses to different ratios of the same two molecules, revealing an efficient mechanism for increasing coding potential of a limited repertoire of molecular signals. The endogenous function of the male sex pheromones has an important side benefit. It substantially ameliorates the detrimental effects of prolonged heat stress on hermaphrodite reproduction because it increases the effectiveness with which surviving gametes are used following stress. Hermaphroditic species are expected to lose female-specific traits in the course of evolution. Our results suggest that some of these traits could have serendipitous utility due to their ability to counter the effects of stress. We propose that this is a general mechanism by which some mating-related functions could be retained in hermaphroditic species, despite their expected decay.

To ensure long-term reproductive success organisms have to cope with harsh environmental extremes. A reproductive strategy that simply maximizes offspring production is likely to be disadvantageous because it could lead to a catastrophic loss of fecundity under unfavorable conditions. To understand how an appropriate balance is achieved, we investigated reproductive performance of C. elegans under conditions of chronic heat stress. We found that following even prolonged exposure to temperatures at which none of the offspring survive, worms could recover and resume reproduction. The likelihood of producing viable offspring falls precipitously after exposure to temperatures greater than 28°C primarily due to sperm damage. Surprisingly, we found that worms that experienced higher temperatures can recover considerably better, provided they did not initiate ovulation. Therefore mechanisms controlling this process must play a crucial role in determining the probability of recovery. We show, however, that suppressing ovulation is only beneficial under relatively long stresses, whereas it is a disadvantageous strategy under shorter stresses of the same intensity. This is because the benefit of shutting down egg laying, and thus protecting the reproductive system, is negated by the cost associated with implementing this strategy--it takes considerable time to recover and produce offspring. We interpret these balanced trade-offs as a dynamic response of the C. elegans reproductive system to stress and an adaptation to life in variable and unpredictable conditions.

Because there is considerable variation in gene expression even between closely related species, it is clear that gene regulatory mechanisms evolve relatively rapidly. Because primary sequence conservation is an unreliable proxy for functional conservation of cis-regulatory elements, their assessment must be carried out in vivo. We conducted a survey of cis-regulatory conservation between C. elegans and closely related species C. briggsae, C. remanei, C. brenneri, and C. japonica. We tested enhancers of eight genes from these species by introducing them into C. elegans and analyzing the expression patterns they drove. Our results support several notable conclusions. Most exogenous cis elements direct expression in the same cells as their C. elegans orthologs, confirming gross conservation of regulatory mechanisms. However, the majority of exogenous elements, when placed in C. elegans, also directed expression in cells outside endogenous patterns, suggesting functional divergence. Recurrent ectopic expression of different promoters in the same C. elegans cells may reflect biases in the directions in which expression patterns can evolve due to shared regulatory logic of coexpressed genes. The fact that, despite differences between individual genes, several patterns repeatedly emerged from our survey, encourages us to think that general rules governing regulatory evolution may exist and be discoverable.

Perennial questions of evolutionary biology can be applied to gene regulatory systems using the abundance of experimental data addressing gene regulation in a comparative context. What is the tempo (frequency, rate) and mode (way, mechanism) of transcriptional regulatory evolution? Here we synthesize the results of 230 experiments performed on insects and nematodes in which regulatory DNA from one species was used to drive gene expression in another species. General principles of regulatory evolution emerge. Gene regulatory evolution is widespread and accumulates with genetic divergence in both insects and nematodes. Divergence in cis is more common than divergence in trans. Coevolution between cis and trans shows a particular increase over greater evolutionary timespans, especially in sex-specific gene regulation. Despite these generalities, the evolution of gene regulation is gene- and taxon-specific. The congruence of these conclusions with evidence from other types of experiments suggests that general principles are discoverable, and a unified view of the tempo and mode of regulatory evolution may be achievable.

Recent studies of animal metabolism have revealed large numbers of novel metabolites that are involved in all aspects of organismal biology, but it is unclear to what extent metabolomes differ between sexes. Here, using untargeted comparative metabolomics for the analysis of wildtype animals and sex determination mutants, we show that C. elegans hermaphrodites and males exhibit pervasive metabolomic differences. Several hundred small molecules are produced exclusively or in much larger amounts in one sex, including a host of previously unreported metabolites that incorporate building blocks from nucleoside, carbohydrate, lipid, and amino acid metabolism. A subset of male-enriched metabolites is specifically associated with the presence of a male germline, whereas enrichment of other compounds requires a male soma. Further, we show that one of the male germline-dependent metabolites, an unusual dipeptide incorporating N , N -dimethyltryptophan, increases food consumption, reduces lifespan, and accelerates the last stage of larval development in hermaphrodites. Our results serve as a foundation for mechanistic studies of how the genetic sex of soma and germline shape the C. elegans metabolome and provide a blueprint for the discovery of sex-dependent metabolites in other animals. Biological sex affects all aspects of animal physiology. Using the model C. elegans , the authors show that metabolomes are highly sex-specific and include a vast space of yet unidentified metabolites that may control development and lifespan.

Natural environments are considerably more variable than laboratory settings and often involve transient exposure to stressful conditions. To fully understand how organisms have evolved to respond to any given stress, prior experience must therefore be considered. We investigated the effects of individual and ancestral experience on C. elegans reproduction. We documented ways in which cultivation at 15°C or 25°C affects developmental time, lifetime fecundity, and reproductive performance after severe heat stress that exceeds the fertile range of the organism but is compatible with survival and future fecundity. We found that experience modulates multiple aspects of reproductive physiology, including the male and female germ lines and the interaction between them. These responses vary in their environmental sensitivity, suggesting the existence of complex mechanisms for coping with unpredictable and stressful environments.

Phenotypes that appear to be conserved could be maintained not only by strong purifying selection on the underlying genetic systems, but also by stabilizing selection acting via compensatory mutations with balanced effects. Such coevolution has been invoked to explain experimental results, but has rarely been the focus of study. Conserved expression driven by the unc-47 promoters of Caenorhabditis elegans and C. briggsae persists despite divergence within a cis-regulatory element and between this element and the trans-regulatory environment. Compensatory changes in cis and trans are revealed when these promoters are used to drive expression in the other species. Functional changes in the C. briggsae promoter, which has experienced accelerated sequence evolution, did not lead to alteration of gene expression in its endogenous environment. Coevolution among promoter elements suggests that complex epistatic interactions within cis-regulatory elements may facilitate their divergence. Our results offer a detailed picture of regulatory evolution in which subtle, lineage-specific, and compensatory modifications of interacting cis and trans regulators together maintain conserved gene expression patterns.

Gene regulatory information guides development and shapes the course of evolution. To test conservation of gene regulation within the phylum Nematoda, we compared the functions of putative cis-regulatory sequences of four sets of orthologs (unc-47, unc-25, mec-3 and elt-2) from distantly-related nematode species. These species, Caenorhabditis elegans, its congeneric C. briggsae, and three parasitic species Meloidogyne hapla, Brugia malayi, and Trichinella spiralis, represent four of the five major clades in the phylum Nematoda. Despite the great phylogenetic distances sampled and the extensive sequence divergence of nematode genomes, all but one of the regulatory elements we tested are able to drive at least a subset of the expected gene expression patterns. We show that functionally conserved cis-regulatory elements have no more extended sequence similarity to their C. elegans orthologs than would be expected by chance, but they do harbor motifs that are important for proper expression of the C. elegans genes. These motifs are too short to be distinguished from the background level of sequence similarity, and while identical in sequence they are not conserved in orientation or position. Functional tests reveal that some of these motifs contribute to proper expression. Our results suggest that conserved regulatory circuitry can persist despite considerable turnover within cis elements.

Different functional constraints contribute to different evolutionary rates across genomes. To understand why some sequences evolve faster than others in a single cis-regulatory locus, we investigated function and evolutionary dynamics of the promoter of the Caenorhabditis elegans unc-47 gene. We found that this promoter consists of two distinct domains. The proximal promoter is conserved and is largely sufficient to direct appropriate spatial expression. The distal promoter displays little if any conservation between several closely related nematodes. Despite this divergence, sequences from all species confer robustness of expression, arguing that this function does not require substantial sequence conservation. We showed that even unrelated sequences have the ability to promote robust expression. A prominent feature shared by all of these robustness-promoting sequences is an AT-enriched nucleotide composition consistent with nucleosome depletion. Because general sequence composition can be maintained despite sequence turnover, our results explain how different functional constraints can lead to vastly disparate rates of sequence divergence within a promoter.