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 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 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.

The Genome Architecture of the Copepod Eurytemora carolleeae, the Highly Invasive Atlantic Clade of the E. affinis Species Complex.

Copepods are among the most abundant organisms on the planet and play critical functions in aquatic ecosystems. Among copepods, populations of the Eurytemora affinis species complex are numerically dominant in many coastal habitats and are food sources for major fisheries. Intriguingly, certain populations possess the unusual capacity to invade novel salinities on rapid time scales. Despite their ecological importance, high-quality genomic resources have been absent for calanoid copepods, limiting our ability to comprehensively dissect the genome architecture underlying the highly invasive and adaptive capacity of certain populations. Here, we presented the first chromosome-level genome of a calanoid copepod, from the Atlantic clade (Eurytemora carolleeae) of the E. affinis species complex. This genome was assembled using high-coverage long-read and high-throughput chromosome conformation capture sequences of an inbred line, generated through 30 generations of full-sib mating. This genome, consisting of 529.3 megabase (Mb) (contig N50 = 4.2 Mb, scaffold N50 = 140.6 Mb), was anchored onto four chromosomes. Genome annotation predicted 20,262 protein-coding genes, of which ion transporter gene families were substantially expanded based on comparative analyses of 12 additional arthropod genomes. Also, we found genome-wide signatures of historical gene body methylation of the ion transporter genes and the significant clustering of these genes on each chromosome. This genome represents one of the most contiguous copepod genomes to date and among the highest quality marine invertebrate genomes. As such, this genome provides an invaluable resource to help yield fundamental insights into the ability of this copepod to adapt to rapidly changing environments.

Evolution of ion transporter Na+/K+-ATPase expression in the osmoregulatory maxillary glands of an invasive copepod.

While many freshwater invaders originate from saline habitats, the physiological mechanisms involved are poorly understood. We investigated the evolution of ion transporter Na+/K+-ATPase (NKA) protein expression between ancestral saline and freshwater invading populations of the copepod Eurytemora carolleae (Atlantic clade of the E. affinis complex). We compared in situ NKA expression between populations under common-garden conditions at three salinities in the maxillary glands. We found the evolution of reduced NKA expression in the freshwater population under freshwater conditions and reduced plasticity (canalization) across salinities, relative to the saline population. Our results support the hypothesis that maxillary glands are involved in ion reabsorption from excretory fluids at low-salinity conditions in the saline population. However, mechanisms of freshwater adaptation, such as increased ion uptake from the environment, might reduce the need for ion reabsorption in the freshwater population. These patterns of ion transporter expression contribute insights into the evolution of ionic regulation during habitat change.

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.

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.

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.