Genome architecture underlying salinity adaptation in the invasive copepod Eurytemora affinis species complex: A review.

With climate change, habitat salinity is shifting rapidly throughout the globe. In addition, many destructive freshwater invaders are recent immigrants from saline habitats. Recently, populations of the copepod Eurytemora affinis species complex have invaded freshwater habitats multiple times independently from saline estuaries on three continents. This review discusses features of this species complex that could enhance their evolutionary potential during rapid environmental change. Remarkably, across independent freshwater invasions, natural selection has repeatedly favored the same alleles far more than expected. This high degree of parallelism is surprising, given the expectation of nonparallel evolution for polygenic adaptation. Factors such as population structure and the genome architecture underlying critical traits under selection might help drive rapid adaptation and parallel evolution. Given the preponderance of saline-to-freshwater invasions and climate-induced salinity change, the principles found here could provide invaluable insights into mechanisms operating in other systems and the potential for adaptation in a changing planet.

Mechanisms of Na+ uptake from freshwater habitats in animals.

Life in fresh water is osmotically and energetically challenging for living organisms, requiring increases in ion uptake from dilute environments. However, mechanisms of ion uptake from freshwater environments are still poorly understood and controversial, especially in arthropods, for which several hypothetical models have been proposed based on incomplete data. One compelling model involves the proton pump V-type H+ ATPase (VHA), which energizes the apical membrane, enabling the uptake of Na+ (and other cations) via an unknown Na+ transporter (referred to as the "Wieczorek Exchanger" in insects). What evidence exists for this model of ion uptake and what is this mystery exchanger or channel that cooperates with VHA? We present results from studies that explore this question in crustaceans, insects, and teleost fish. We argue that the Na+/H+ antiporter (NHA) is a likely candidate for the Wieczorek Exchanger in many crustaceans and insects; although, there is no evidence that this is the case for fish. NHA was discovered relatively recently in animals and its functions have not been well characterized. Teleost fish exhibit redundancy of Na+ uptake pathways at the gill level, performed by different ion transporter paralogs in diverse cell types, apparently enabling tolerance of low environmental salinity and various pH levels. We argue that much more research is needed on overall mechanisms of ion uptake from freshwater habitats, especially on NHA and other potential Wieczorek Exchangers. Such insights gained would contribute greatly to our general understanding of ionic regulation in diverse species across habitats.

Genome-wide signatures of synergistic epistasis during parallel adaptation in a Baltic Sea copepod.

The role of epistasis in driving adaptation has remained an unresolved problem dating back to the Evolutionary Synthesis. In particular, whether epistatic interactions among genes could promote parallel evolution remains unexplored. To address this problem, we employ an Evolve and Resequence (E&R) experiment, using the copepod Eurytemora affinis, to elucidate the evolutionary genomic response to rapid salinity decline. Rapid declines in coastal salinity at high latitudes are a predicted consequence of global climate change. Based on time-resolved pooled whole-genome sequencing, we uncover a remarkably parallel, polygenic response across ten replicate selection lines, with 79.4% of selected alleles shared between lines by the tenth generation of natural selection. Using extensive computer simulations of our experiment conditions, we find that this polygenic parallelism is consistent with positive synergistic epistasis among alleles, far more so than other mechanisms tested. Our study provides experimental and theoretical support for a novel mechanism promoting repeatable polygenic adaptation, a phenomenon that may be common for selection on complex physiological traits.

Recognizing Salinity Threats in the Climate Crisis.

Climate change is causing habitat salinity to transform at unprecedented rates across the globe. While much of the research on climate change has focused on rapid shifts in temperature, far less attention has focused on the effects of changes in environmental salinity. Consequently, predictive studies on the physiological, evolutionary, and migratory responses of organisms and populations to the threats of salinity change are relatively lacking. This omission represents a major oversight, given that salinity is among the most important factors that define biogeographic boundaries in aquatic habitats. In this perspective, we briefly touch on responses of organisms and populations to rapid changes in salinity occurring on contemporary time scales. We then discuss factors that might confer resilience to certain taxa, enabling them to survive rapid salinity shifts. Next, we consider approaches for predicting how geographic distributions will shift in response to salinity change. Finally, we identify additional data that are needed to make better predictions in the future. Future studies on climate change should account for the multiple environmental factors that are rapidly changing, especially habitat salinity.

Ion Transporter Gene Families as Physiological Targets of Natural Selection During Salinity Transitions in a Copepod.

Salinity is a key factor that structures biodiversity on the planet. With anthropogenic change, such as climate change and species invasions, many populations are facing rapid and dramatic changes in salinity throughout the globe. Studies on the copepod Eurytemora affinis species complex have implicated ion transporter gene families as major loci contributing to salinity adaptation during freshwater invasions. Laboratory experiments and population genomic surveys of wild populations have revealed evolutionary shifts in genome-wide gene expression and parallel genomic signatures of natural selection during independent salinity transitions. Our results suggest that balancing selection in the native range and epistatic interactions among specific ion transporter paralogs could contribute to parallel freshwater adaptation. Overall, these studies provide unprecedented insights into evolutionary mechanisms underlying physiological adaptation during rapid salinity change.

Rapid evolution of genome-wide gene expression and plasticity during saline to freshwater invasions by the copepod Eurytemora affinis species complex.

Saline migrants into freshwater habitats constitute among the most destructive invaders in aquatic ecosystems throughout the globe. However, the evolutionary and physiological mechanisms underlying such habitat transitions remain poorly understood. To explore the mechanisms of freshwater adaptation and distinguish between adaptive (evolutionary) and acclimatory (plastic) responses to salinity change, we examined genome-wide patterns of gene expression between ancestral saline and derived freshwater populations of the Eurytemora affinis species complex, reared under two different common-garden conditions (0 versus 15 PSU). We found that evolutionary shifts in gene expression (between saline and freshwater inbred lines) showed far greater changes and were more widespread than acclimatory responses to salinity (0 versus 15 PSU). Most notably, 30-40 genes showing evolutionary shifts in gene expression across the salinity boundary were associated with ion transport function, with inorganic cation transmembrane transport forming the largest Gene Ontology category. Of particular interest was the sodium transporter, the Na+ /H+ antiporter (NHA) gene family, which was discovered in animals relatively recently. Thirty key ion regulatory genes, such as NHA paralogue #7, demonstrated concordant evolutionary and plastic shifts in gene expression, suggesting the evolution of ion transporter function and plasticity during rapid invasions into novel salinities. Moreover, freshwater invasions were associated with the evolution of reduced plasticity in the freshwater population, again for the same key ion transporters, consistent with the predicted evolution of canalization following adaptation to stressful conditions. Our results have important implications for understanding evolutionary and physiological mechanisms of range expansions by some of the most widespread invaders in aquatic habitats.

Evolutionary origins of genomic adaptations in an invasive copepod.

The ability of populations to expand their geographical ranges, whether as invaders, agricultural strains or climate migrants, is currently one of the most serious global problems. However, fundamental mechanisms remain poorly understood regarding factors that enable certain populations, such as biological invaders, to rapidly transition to novel habitats. According to one hypothesis, environmental fluctuations in the native range could promote successful invasions by imposing balancing selection on key traits and maintaining the genetic variation that enables rapid adaptation in novel habitats. Here we test the genomic predictions of this hypothesis by performing whole-genome sequencing of multiple independent invasive freshwater and native saline populations of the copepod Eurytemora affinis complex. We found that invasive populations have repeatedly responded to selection through the parallel use of the same single-nucleotide polymorphisms and genomic loci, to a much greater degree than expected. These same loci were enriched for signatures of long-term balancing selection in the native ranges, with 15-47% of loci exhibiting significant signatures of balancing selection. The strong association between parallel evolution in the invaded range and balancing selection in the native range supports the hypothesis that fluctuating habitats can promote invasive success and that balancing selection might serve as a widespread and important mechanism that enables rapid adaptation in nature.

Gene content evolution in the arthropods.

BACKGROUND: Arthropods comprise the largest and most diverse phylum on Earth and play vital roles in nearly every ecosystem. Their diversity stems in part from variations on a conserved body plan, resulting from and recorded in adaptive changes in the genome. Dissection of the genomic record of sequence change enables broad questions regarding genome evolution to be addressed, even across hyper-diverse taxa within arthropods. RESULTS: Using 76 whole genome sequences representing 21 orders spanning more than 500 million years of arthropod evolution, we document changes in gene and protein domain content and provide temporal and phylogenetic context for interpreting these innovations. We identify many novel gene families that arose early in the evolution of arthropods and during the diversification of insects into modern orders. We reveal unexpected variation in patterns of DNA methylation across arthropods and examples of gene family and protein domain evolution coincident with the appearance of notable phenotypic and physiological adaptations such as flight, metamorphosis, sociality, and chemoperception. CONCLUSIONS: These analyses demonstrate how large-scale comparative genomics can provide broad new insights into the genotype to phenotype map and generate testable hypotheses about the evolution of animal diversity.

The Toxicogenome of Hyalella azteca: A Model for Sediment Ecotoxicology and Evolutionary Toxicology.

Hyalella azteca is a cryptic species complex of epibenthic amphipods of interest to ecotoxicology and evolutionary biology. It is the primary crustacean used in North America for sediment toxicity testing and an emerging model for molecular ecotoxicology. To provide molecular resources for sediment quality assessments and evolutionary studies, we sequenced, assembled, and annotated the genome of the H. azteca U.S. Lab Strain. The genome quality and completeness is comparable with other ecotoxicological model species. Through targeted investigation and use of gene expression data sets of H. azteca exposed to pesticides, metals, and other emerging contaminants, we annotated and characterized the major gene families involved in sequestration, detoxification, oxidative stress, and toxicant response. Our results revealed gene loss related to light sensing, but a large expansion in chemoreceptors, likely underlying sensory shifts necessary in their low light habitats. Gene family expansions were also noted for cytochrome P450 genes, cuticle proteins, ion transporters, and include recent gene duplications in the metal sequestration protein, metallothionein. Mapping of differentially expressed transcripts to the genome significantly increased the ability to functionally annotate toxicant responsive genes. The H. azteca genome will greatly facilitate development of genomic tools for environmental assessments and promote an understanding of how evolution shapes toxicological pathways with implications for environmental and human health.

Evolutionary responses to crude oil from the Deepwater Horizon oil spill by the copepod Eurytemora affinis.

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.

Evolutionary History of Chemosensory-Related Gene Families across the Arthropoda.

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 Legs Have It: In Situ Expression of Ion Transporters V-Type H(+)-ATPase and Na(+)/K(+)-ATPase in the Osmoregulatory Leg Organs of the Invading Copepod Eurytemora affinis.

The copepod Eurytemora affinis has an unusually broad salinity range, as some populations have recently invaded freshwater habitats independently from their ancestral saline habitats. Prior studies have shown evolutionary shifts in ion transporter activity during freshwater invasions and localization of ion transporters in newly discovered "Crusalis organs" in the swimming legs. The goals of this study were to localize and quantify expression of ion transport enzymes V-type H(+)-ATPase (VHA) and Na(+)/K(+)-ATPase (NKA) in the swimming legs of E. affinis and determine the degree of involvement of each leg in ionic regulation. We confirmed the presence of two distinct types of ionocytes in the Crusalis organs. Both cell types expressed VHA and NKA, and in the freshwater population the location of VHA and NKA in ionocytes was, respectively, apical and basal. Quantification of in situ expression of NKA and VHA established the predominance of swimming leg pairs 3 and 4 in ion transport in both saline and freshwater populations. Increases in VHA expression in swimming legs 3 and 4 of the freshwater population (in fresh water) relative to the saline population (at 15 PSU) arose from an increase in the abundance of VHA per cell rather than an increase in the number of ionocytes. This result suggests a simple mechanism for increasing ion uptake in fresh water. In contrast, the decline in NKA expression in the freshwater population arose from a decrease in ionocyte area in legs 4, likely resulting from decreases in number or size of ionocytes containing NKA. Such results provide insights into mechanisms of ionic regulation for this species, with added insights into evolutionary mechanisms underlying physiological adaptation during habitat invasions.

Evolutionary mechanisms of habitat invasions, using the copepod Eurytemora affinis as a model system.

The study of the copepod Eurytemora affinis has provided unprecedented insights into mechanisms of invasive success. In this invited review, I summarize a subset of work from my laboratory to highlight key insights gained from studying E. affinis as a model system. Invasive species with brackish origins are overrepresented in freshwater habitats. The copepod E. affinis is an example of such a brackish invader, and has invaded freshwater habitats multiple times independently in recent years. These invasions were accompanied by the evolution of physiological tolerance and plasticity, increased body fluid regulation, and evolutionary shifts in ion transporter (V-type H(+) ATPase, Na(+), K(+)-ATPase) activity and expression. These evolutionary changes occurred in parallel across independent invasions in nature and in laboratory selection experiments. Selection appears to act on standing genetic variation during invasions, and maintenance of this variation is likely facilitated through 'beneficial reversal of dominance' in salinity tolerance across habitats. Expression of critical ion transporters is localized in newly discovered Crusalis leg organs. Increased freshwater tolerance is accompanied by costs to development time and greater requirements for food. High-food concentration increases low-salinity tolerance, allowing saline populations to invade freshwater habitats. Mechanisms observed here likely have relevance for other taxa undergoing fundamental niche expansions.

Testing for beneficial reversal of dominance during salinity shifts in the invasive copepod Eurytemora affinis, and implications for the maintenance of genetic variation.

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 saltwater-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.

Without gills: localization of osmoregulatory function in the copepod Eurytemora affinis.

The Pancrustacea, which include crustaceans and hexapods, have successfully colonized marine, freshwater, and terrestrial habitats. While members of the class Malacostraca (e.g., crabs, shrimp) often display immense osmoregulatory capacities, more basally branching crustaceans (e.g., copepods, branchiopods) tend to possess less-specialized osmoregulatory structures that have been poorly characterized. Remarkably, some of these more basal taxa have also colonized diverse habitats. For instance, the copepod Eurytemora affinis has recently invaded freshwater habitats multiple times independently but lack obvious osmoregulatory structures. To explore localization of ion exchange, we performed silver staining, immunohistochemical staining, and transmission electron microscopy. Our results revealed localization of ion transport within the maxillary glands and on four pairs of swimming legs. Silver staining revealed ion exchange at the maxillary pores and on the endopods and exopods of swimming legs P1 through P4. Immunohistochemical assays localized ion transport enzymes V-type H(+)-ATPase and Na(+)/K(+)-ATPase in the maxillary glands and swimming legs as well. Finally, transmission electron microscopy identified specialized ionocytes within these anatomical regions. These investigations uncovered novel osmoregulatory structures at the swimming legs, which we designate the "Crusalis organs." Our findings identified specific tissues specialized for ion transport, potentially enabling this small crustacean to rapidly transition into freshwater habitats.

Feasting in fresh water: impacts of food concentration on freshwater tolerance and the evolution of food × salinity response during the expansion from saline into fresh water habitats.

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.

Direct sequencing of haplotypes from diploid individuals through a modified emulsion PCR-based single-molecule sequencing approach.

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, 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.

Rapid evolution of body fluid regulation following independent invasions into freshwater habitats.

Colonizations from marine to freshwater environments constitute among the most dramatic evolutionary transitions in the history of life. Colonizing dilute environments poses great challenges for acquiring essential ions against steep concentration gradients. This study explored the evolution of body fluid regulation following freshwater invasions by the copepod Eurytemora affinis. The goals of this study were to determine (1) whether invasions from saline to freshwater habitats were accompanied by evolutionary shifts in body fluid regulation (hemolymph osmolality) and (2) whether parallel shifts occurred during independent invasions. We measured hemolymph osmolality for ancestral saline and freshwater invading populations reared across a range of common-garden salinities (0.2-25 PSU). Our results revealed the evolution of increased hemolymph osmolality (by 16-31%) at lower salinities in freshwater populations of E. affinis relative to their saline ancestors. Moreover, we observed the same evolutionary shifts across two independent freshwater invasions. Such increases in hemolymph osmolality are consistent with evidence of increased ion uptake in freshwater populations at low salinity, found in a previous study, and are likely to entail increased energetic costs upon invading freshwater habitats. Our findings are consistent with the evolution of increased physiological regulation accompanying transitions into stressful environments.

Observing copepods through a genomic lens.

BACKGROUND: Copepods outnumber every other multicellular animal group. They are critical components of the world's freshwater and marine ecosystems, sensitive indicators of local and global climate change, key ecosystem service providers, parasites and predators of economically important aquatic animals and potential vectors of waterborne disease. Copepods sustain the world fisheries that nourish and support human populations. Although genomic tools have transformed many areas of biological and biomedical research, their power to elucidate aspects of the biology, behavior and ecology of copepods has only recently begun to be exploited. DISCUSSION: The extraordinary biological and ecological diversity of the subclass Copepoda provides both unique advantages for addressing key problems in aquatic systems and formidable challenges for developing a focused genomics strategy. This article provides an overview of genomic studies of copepods and discusses strategies for using genomics tools to address key questions at levels extending from individuals to ecosystems. Genomics can, for instance, help to decipher patterns of genome evolution such as those that occur during transitions from free living to symbiotic and parasitic lifestyles and can assist in the identification of genetic mechanisms and accompanying physiological changes associated with adaptation to new or physiologically challenging environments. The adaptive significance of the diversity in genome size and unique mechanisms of genome reorganization during development could similarly be explored. Genome-wide and EST studies of parasitic copepods of salmon and large EST studies of selected free-living copepods have demonstrated the potential utility of modern genomics approaches for the study of copepods and have generated resources such as EST libraries, shotgun genome sequences, BAC libraries, genome maps and inbred lines that will be invaluable in assisting further efforts to provide genomics tools for copepods. SUMMARY: Genomics research on copepods is needed to extend our exploration and characterization of their fundamental biological traits, so that we can better understand how copepods function and interact in diverse environments. Availability of large scale genomics resources will also open doors to a wide range of systems biology type studies that view the organism as the fundamental system in which to address key questions in ecology and evolution.

Pumping ions: rapid parallel evolution of ionic regulation following habitat invasions.

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.