Chemosensory-related gene (CRG) families have been studied extensively in insects, but their evolutionary history across the Arthropoda had remained relatively unexplored. Here, we address current hypotheses and prior conclusions on CRG family evolution using a more comprehensive data set. In particular, odorant receptors were hypothesized to have proliferated during terrestrial colonization by insects (hexapods), but their association with other pancrustacean clades and with independent terrestrial colonizations in other arthropod subphyla have been unclear. We also examine hypotheses on which arthropod CRG family is most ancient. Thus, we reconstructed phylogenies of CRGs, including those from new arthropod genomes and transcriptomes, and mapped CRG gains and losses across arthropod lineages. Our analysis was strengthened by including crustaceans, especially copepods, which reside outside the hexapod/branchiopod clade within the subphylum Pancrustacea. We generated the first high-resolution genome sequence of the copepod Eurytemora affinis and annotated its CRGs. We found odorant receptors and odorant binding proteins present only in hexapods (insects) and absent from all other arthropod lineages, indicating that they are not universal adaptations to land. Gustatory receptors likely represent the oldest chemosensory receptors among CRGs, dating back to the Placozoa. We also clarified and confirmed the evolutionary history of antennal ionotropic receptors across the Arthropoda. All antennal ionotropic receptors in E. affinis were expressed more highly in males than in females, suggestive of an association with male mate-recognition behavior. This study is the most comprehensive comparative analysis to date of CRG family evolution across the largest and most speciose metazoan phylum Arthropoda.

The BP Deepwater Horizon Oil Disaster was the most catastrophic offshore oil spill in U.S. history, yet we still have a poor understanding of how organisms could evolve in response to the toxic effects of crude oil. This study offers a rare analysis of how fitness-related traits could evolve rapidly in response to crude oil toxicity. We examined evolutionary responses of populations of the common copepod Eurytemora affinis residing in the Gulf of Mexico, by comparing crude oil tolerance of populations collected before versus after the Deepwater Horizon oil spill of 2010. In addition, we imposed laboratory selection for crude oil tolerance for ~8 generations, using an E. affinis population collected from before the oil spill. We found evolutionary increases in crude oil tolerance in the wild population following the oil spill, relative to the population collected before the oil spill. The post-oil spill population showed increased survival and rapid development time in the presence of crude oil. In contrast, evolutionary responses following laboratory selection were less clear; though, development time from metamorphosis to adult in the presence of crude oil did become more rapid after selection. We did find that the wild population, used in both experiments, harbored significant genetic variation in crude oil tolerance, upon which selection could act. Thus, our study indicated that crude oil tolerance could evolve, but perhaps not on the relatively short time scale of the laboratory selection experiment. This study contributes novel insights into evolutionary responses to crude oil, in directly examining fitness-related traits before and after an oil spill, and in observing evolutionary responses following laboratory selection.

Maintenance of genetic variation at loci under selection has profound implications for adaptation under environmental change. In temporally and spatially varying habitats, non-neutral polymorphism could be maintained by heterozygote advantage across environments (marginal overdominance), which could be greatly increased by beneficial reversal of dominance across conditions. We tested for reversal of dominance and marginal overdominance in salinity tolerance in the salt-to-freshwater invading copepod Eurytemora affinis. We compared survival of F1 offspring generated by crossing saline and freshwater inbred lines (between-salinity F1 crosses) relative to within-salinity F1 crosses, across three salinities. We found evidence for both beneficial reversal of dominance and marginal overdominance in salinity tolerance. In support of reversal of dominance, survival of between-salinity F1 crosses was not different from that of freshwater F1 crosses under freshwater conditions and saltwater F1 crosses under saltwater conditions. In support of marginal overdominance, between-salinity F1 crosses exhibited significantly higher survival across salinities relative to both freshwater and saltwater F1 crosses. Our study provides a rare empirical example of complete beneficial reversal of dominance associated with environmental change. This mechanism might be crucial for maintaining genetic variation in salinity tolerance in E. affinis populations, allowing rapid adaptation to salinity changes during habitat invasions.

Saline to freshwater invasions have become increasingly common in recent years. A key hypothesis is that rates of freshwater invasions have been amplified in recent years by increased food concentration, yet this hypothesis has remained unexplored. We examined whether elevated food concentration could enhance freshwater tolerance, and whether this effect evolves following saline to freshwater invasions. We examined physiological response to salinity and food concentration in a 2 × 2 factorial design, using ancestral brackish and freshwater invading populations of the copepod Eurytemora affinis. We found that high food concentration significantly increases low-salinity tolerance. This effect was reduced in the freshwater population, indicating evolution following the freshwater invasion. Thus, ample food could enable freshwater invasions, allowing subsequent evolution of low-salinity tolerance even under food-poor conditions. We also compared effects of food concentration on freshwater survival between two brackish populations from the native range. Impacts of food concentration on freshwater survival differed between the brackish populations, suggesting variation in functional properties affecting their propensity to invade freshwater habitats. The key implication is that high food concentration could profoundly extend range expansions of brackishwater species into freshwater habitats, potentially allowing for condition-specific competition between saline invaders and resident freshwater species.

While standard DNA sequencing approaches readily yield genotypic sequence data, haplotype information is often of greater utility for population genetic analyses. However, obtaining individual haplotype sequences can be costly and time consuming, and sometimes requires statistical reconstruction approaches that are subject to bias and error. Advancements have recently been made in determining individual chromosomal sequences in large-scale genomic studies, yet few options exist for obtaining this information from large numbers of highly polymorphic individuals in a cost effective manner. As a solution, we developed a simple PCR-based method for obtaining sequence information from individual DNA strands using standard laboratory equipment. The method employs a water-in-oil emulsion to separate the PCR mixture into thousands of individual microreactors. PCR within these small vesicles results in amplification from only a single starting DNA template molecule, and thus a single haplotype. We improved upon previous approaches by including SYBR Green I and a melted agarose solution in the PCR reaction, allowing easy identification and separation of individually amplified DNA molecules. We demonstrate the use of this method on a highly polymorphic estuarine population of the copepod Eurytemora affinis for which current molecular and computational methods for haplotype determination have been inadequate.

Marine to freshwater colonizations constitute among the most dramatic evolutionary transitions in the history of life. This study examined evolution of ionic regulation following saline-to-freshwater transitions in an invasive species. In recent years, the copepod Eurytemora affinis has invaded freshwater habitats multiple times independently. We found parallel evolutionary shifts in ion-motive enzyme activity (V-type H+ ATPase, Na+/K+-ATPase) across independent invasions and in replicate laboratory selection experiments. Freshwater populations exhibited increased V-type H+ ATPase activity in fresh water (0 PSU) and declines at higher salinity (15 PSU) relative to saline populations. This shift represented marked evolutionary increases in plasticity. In contrast, freshwater populations displayed reduced Na+/K+-ATPase activity across all salinities. Most notably, modifying salinity alone during laboratory selection experiments recapitulated the evolutionary shifts in V-type H+ ATPase activity observed in nature. Maternal and embryonic acclimation could not account for the observed shifts in enzyme activity. V-type H+ ATPase function has been hypothesized to be critical for freshwater and terrestrial adaptations, but evolution of this enzyme function had not been previously demonstrated in the context of habitat transitions. Moreover, the speed of these evolutionary shifts was remarkable, within a few generations in the laboratory and a few decades in the wild.