Local adaptation and future climate vulnerability in a wild rodent.

As climate change continues, species pushed outside their physiological tolerance limits must adapt or face extinction. When change is rapid, adaptation will largely harness ancestral variation, making the availability and characteristics of that variation of critical importance. Here, we used whole-genome sequencing and genetic-environment association analyses to identify adaptive variation and its significance in the context of future climates in a small Palearctic mammal, the bank vole (Clethrionomys glareolus). We found that peripheral populations of bank vole in Britain are already at the extreme bounds of potential genetic adaptation and may require an influx of adaptive variation in order to respond. Analyses of adaptive loci suggest regional differences in climate variables select for variants that influence patterns of population adaptive resilience, including genes associated with antioxidant defense, and support a pattern of thermal/hypoxic cross-adaptation. Our findings indicate that understanding potential shifts in genomic composition in response to climate change may be key to predicting species' fate under future climates.

Resurrection genomics provides molecular and phenotypic evidence of rapid adaptation to salinization in a keystone aquatic species.

Ecologists and evolutionary biologists are increasingly cognizant of rapid adaptation in wild populations. Rapid adaptation to anthropogenic environmental change is critical for maintaining biodiversity and ecosystems services into the future. Anthropogenic salinization of freshwater ecosystems is quickly emerging as a primary threat, which is well documented in the northern temperate ecoregion. Specifically, many northern temperate lakes have undergone extensive salinization because of urbanization and the associated increase in impervious surfaces causing runoff, and the extensive use of road deicing salts (e.g., NaCl). It remains unclear whether increasing salinization will lead to extirpation of species from these systems. Using a "resurrection genomics" approach, we investigated whether the keystone aquatic herbivore, Daphnia pulicaria, has evolved increased salinity tolerance in a severely salinized lake located in Minnesota, USA. Whole-genome resequencing of 54 Daphnia clones from the lake and hatched from resting eggs that represent a 25-y temporal contrast demonstrates that many regions of the genome containing genes related to osmoregulation are under selection in the study population. Tolerance assays of clones revealed that the most recent clones are more tolerant to salinity than older clones; this pattern is concomitant with the temporal pattern of stabilizing salinity in this lake. Together, our results demonstrate that keystone species such as Daphnia can rapidly adapt to increasing freshwater salinization. Further, our results indicate that rapid adaptation to salinity may allow lake Daphnia populations to persist in the face of anthropogenic salinization maintaining the food webs and ecosystem services they support despite global environmental change.

The roles of recombination and selection in shaping genomic divergence in an incipient ecological species complex.

Speciation genomic studies have revealed that genomes of diverging lineages are shaped jointly by the actions of gene flow and selection. These evolutionary forces acting in concert with processes such as recombination and genome features such as gene density shape a mosaic landscape of divergence. We investigated the roles of recombination and gene density in shaping the patterns of differentiation and divergence between the cyclically parthenogenetic ecological sister-taxa, Daphnia pulicaria and Daphnia pulex. First, we assembled a phased chromosome-scale genome assembly using trio-binning for D. pulicaria and constructed a genetic map using an F2-intercross panel to understand sex-specific recombination rate heterogeneity. Finally, we used a ddRADseq data set with broad geographic sampling of D. pulicaria, D. pulex, and their hybrids to understand the patterns of genome-scale divergence and demographic parameters. Our study provides the first sex-specific estimates of recombination rates for a cyclical parthenogen, and unlike other eukaryotic species, we observed male-biased heterochiasmy in D. pulicaria, which may be related to this somewhat unique breeding mode. Additionally, regions of high gene density and recombination are generally more divergent than regions of suppressed recombination. Outlier analysis indicated that divergent genomic regions are probably driven by selection on D. pulicaria, the derived lineage colonizing a novel lake habitat. Together, our study supports a scenario of selection acting on genes related to local adaptation shaping genome-wide patterns of differentiation despite high local recombination rates in this species complex. Finally, we discuss the limitations of our data in light of demographic uncertainty.

Quantitative genetics of phosphorus content in the freshwater herbivore, Daphnia pulicaria.

Phosphorus (P) is essential for growth of all organisms, and P content is correlated with growth in most taxa. Although P content was initially considered to be a trait fixed at the species level, there is growing evidence for considerable intraspecific variation. Selection on such variation can thus alter the rates at which P fluxes through food webs. Nevertheless, prior work describing the sources and extent of intraspecific variation in P content were not genetically explicit, confounded by unknown genetic background and evolutionary history. We constructed an F2 recombinant population of the dominant freshwater grazer, Daphnia pulicaria to mitigate such issues. F2 recombinants exhibited considerable variation in growth rate, P content (0.49%-1.97%), P use efficiency (PUE; 51-208 mg biomass/mg P), and correlated traits such as hatching time of resting eggs, in common garden conditions. These results clearly demonstrate the scope of genetic recombination in generating variation in ecologically relevant traits. The absence of environmental selection is a likely component driving such variation not observed in natural settings. Although phosphoglucose isomerase (PGI) genotype was significantly associated with variation in hatching time of resting eggs, contrary to prior work with less rigorous designs, and allelic variation at the PGI locus did not explain variation in P content and PUE of Daphnia, indicating that such quantitative traits are under polygenic control. Together, these results suggest that although there is considerable genetic scope for variation in key ecologically relevant traits, such as P content and efficiency of P use, these traits are likely under strong stabilizing selection, most likely due to selection on growth rate and size. Importantly, our observations suggest that anthropogenic alterations to P supply due to eutrophication could alter selection on these traits, thereby rapidly altering the role Daphnia plays in the P cycle of lakes.

High genomic diversity in the bank vole at the northern apex of a range expansion: The role of multiple colonizations and end-glacial refugia.

The history of repeated northern glacial cycling and southern climatic stability has long dominated explanations for how genetic diversity is distributed within temperate species in Eurasia and North America. However, growing evidence indicates the importance of cryptic refugia for northern colonization dynamics. An important geographic region to assess this is Fennoscandia, where recolonization at the end of the last glaciation was restricted to specific routes and temporal windows. We used genomic data to analyse genetic diversity and colonization history of the bank vole (Myodes glareolus) throughout Europe (>800 samples) with Fennoscandia as the northern apex. We inferred that bank voles colonized Fennoscandia multiple times by two different routes; with three separate colonizations via a southern land-bridge route deriving from a "Carpathian" glacial refugium and one via a north-eastern route from an "Eastern" glacial refugium near the Ural Mountains. Clustering of genome-wide SNPs revealed high diversity in Fennoscandia, with eight genomic clusters: three of Carpathian origin and five Eastern. Time estimates revealed that the first of the Carpathian colonizations occurred before the Younger Dryas (YD), meaning that the first colonists survived the YD in Fennoscandia. Results also indicated that introgression between bank and northern red-backed voles (Myodes rutilus) took place in Fennoscandia just after end-glacial colonization. Therefore, multiple colonizations from the same and different cryptic refugia, temporal and spatial separations and interspecific introgression have shaped bank vole genetic variability in Fennoscandia. Together, these processes drive high genetic diversity at the apex of the northern expansion in this emerging model species.

Phototactic behavior of native Daphnia in the presence of chemical cues from a non-native predator Bythotrephes.

Chemical cues are used by many taxa to communicate within and among species. Behavioral defenses induced by predator cues are a mechanism by which prey species resist or avoid predator attack. This study examined the egg bank of native Daphnia species in a lake that has been invaded by Bythotrephes longimanus, an invertebrate zooplanktivore native to northern-central Europe and Asia (initial invasion 1994, population boom in 2009). Daphnia resting eggs from both pre- and post-B. longimanus invasion lake sediments were hatched and established as isofemale clonal lines. Phototactic behavior (a proxy for vertical migration behavior) was assessed in the presence and absence of B. longimanus cue. This was done to evaluate the hypothesis that the heavy predation imposed by B. longimanus would have been selected for Daphnia clones that are more negatively phototactic in the presence of B. longimanus cue, because B. longimanus is a visual predator. The behavior of the clones derived from pre-B. longimanus era resting eggs was not significantly different from the behavior of the clones from the post-B. longimanus era and exposure to predator cue did not affect the phototactic response of the clones. There was a significant difference in the phototactic behavior of the three Daphnia species tested (Daphnia ambigua, Daphnia mendotae, and Daphnia pulicaria). These results suggest that predation by B. longimanus is not the main factor that is influencing the phototactic behavior of Daphnia in the lake. Other factors such as fish predation may be playing a more significant role in this system.

Resurrected 'ancient' Daphnia genotypes show reduced thermal stress tolerance compared to modern descendants.

Understanding how populations adapt to rising temperatures has been a challenge in ecology. Research often evaluates multiple populations to test whether local adaptation to temperature regimes is occurring. Space-for-time substitutions are common, as temporal constraints limit our ability to observe evolutionary responses. We employed a resurrection ecology approach to understand how thermal tolerance has changed in a Daphnia pulicaria population over time. Temperatures experienced by the oldest genotypes were considerably lower than the youngest. We hypothesized clones were adapted to the thermal regimes of their respective time periods. We performed two thermal shock experiments that varied in length of heat exposure. Overall trends revealed that younger genotypes exhibited higher thermal tolerance than older genotypes; heat shock protein (hsp70) expression increased with temperature and varied among genotypes, but not across time periods. Our results indicate temperature may have been a selective factor on this population, although the observed responses may be a function of multifarious selection. Prior work found striking changes in population genetic structure, and in other traits that were strongly correlated with anthropogenic changes. Resurrection ecology approaches should help our understanding of interactive effects of anthropogenic alterations to temperature and other stressors on the evolutionary fate of natural populations.

Evolutionary aspects of resurrection ecology: Progress, scope, and applications-An overview.

This perspective provides an overview to the Special Issue on Resurrection Ecology (RE). It summarizes the contributions to this Special Issue, and provides background information and future prospects for the use of RE in both basic and applied evolutionary studies.

Whole genome amplification and sequencing of a Daphnia resting egg.

Resting eggs banks are unique windows that allow us to directly observe shifts in population genetics, and phenotypes over time as natural populations evolve. Though a variety of planktonic organisms also produce resting stages, the keystone freshwater consumer, Daphnia, is a well-known model for paleogenetics and resurrection ecology. Nevertheless, paleogenomic investigations are limited largely because resting eggs do not contain enough DNA for genomic sequencing. In fact, genomic studies even on extant populations include a laborious preparatory phase of batch culturing dozens of individuals to generate sufficient genomic DNA. Here, we furnish a protocol to generate whole genomes of single ephippial (resting) eggs and single daphniids. Whole genomes of single ephippial eggs and single adults were amplified using Qiagen REPLI-g Single Cell kit reaction, followed by NEBNext Ultra DNA Library Prep Kit for library construction and Illumina sequencing. We compared the quality of the single-egg and single-individual amplified genomes to the standard batch genomic DNA extraction in the absence of genome amplification. At mean 20× depth, coverage was essentially identical for the amplified single individual relative to the unamplified batch extracted genome (>90% of the genome was covered and callable). Finally, while amplification resulted in the slight loss of heterozygosity for the amplified genomes, estimates were largely comparable and illustrate the utility and limitations of this approach in estimating population genetic parameters over long periods of time in natural populations of Daphnia and also other small species known to produce resting stages.

Genotype-specific relationships among phosphorus use, growth and abundance in Daphnia pulicaria.

The framework ecological stoichiometry uses elemental composition of species to make predictions about growth and competitive ability in defined elemental supply conditions. Although intraspecific differences in stoichiometry have been observed, we have yet to understand the mechanisms generating and maintaining such variation. We used variation in phosphorus (P) content within a Daphnia species to test the extent to which %P can explain variation in growth and competition. Further, we measured 33P kinetics (acquisition, assimilation, incorporation and retention) to understand the extent to which such variables improved predictions. Genotypes showed significant variation in P content, 33P kinetics and growth rate. P content alone was a poor predictor of growth rate and competitive ability. While most genotypes exhibited the typical growth penalty under P limitation, a few varied little in growth between P diets. These observations indicate that some genotypes can maintain growth under P-limited conditions by altering P use, suggesting that decomposing P content of an individual into physiological components of P kinetics will improve stoichiometric models. More generally, attention to the interplay between nutrient content and nutrient-use is required to make inferences regarding the success of genotypes in defined conditions of nutrient supply.

Endopolyploidy as a potential driver of animal ecology and evolution.

Endopolyploidy - the existence of higher-ploidy cells within organisms that are otherwise of a lower ploidy level (generally diploid) - was discovered decades ago, but remains poorly studied relative to other genomic phenomena, especially in animals. Our synthetic review suggests that endopolyploidy is more common in animals than often recognized and probably influences a number of fitness-related and ecologically important traits. In particular, we argue that endopolyploidy is likely to play a central role in key traits such as gene expression, body and cell size, and growth rate, and in a variety of cell types, including those responsible for tissue regeneration, nutrient storage, and inducible anti-predator defences. We also summarize evidence for intraspecific genetic variation in endopolyploid levels and make the case that the existence of this variation suggests that endopolyploid levels are likely to be heritable and thus a potential target for natural selection. We then discuss why, in light of evident benefits of endopolyploidy, animals remain primarily diploid. We conclude by highlighting key areas for future research such as comprehensive evaluation of the heritability of endopolyploidy and the adaptive scope of endopolyploid-related traits, the extent to which endopolyploid induction incurs costs, and characterization of the relationships between environmental variability and endopolyploid levels.

Paleogenetic records of Daphnia pulicaria in two North American lakes reveal the impact of cultural eutrophication.

Understanding the evolutionary consequences of the green revolution, particularly in wild populations, is an important frontier in contemporary biology. Because human impacts have occurred at varying magnitudes or time periods depending on the study ecosystem, evolutionary histories may vary considerably among populations. Paleogenetics in conjunction with paleolimnology enable us to associate microevolutionary dynamics with detailed information on environmental change. We used this approach to reconstruct changes in the temporal population genetic structure of the keystone zooplankton grazer, Daphnia pulicaria, using dormant eggs extracted from sediments in two Minnesota lakes (South Center, Hill). The extent of agriculture and human population density in the catchment of these lakes has differed markedly since European settlement in the late 19th century and is reflected in their environmental histories reconstructed here. The reconstructed environments of these two lakes differed strongly in terms of environmental stability and their associated patterns of Daphnia population structure. We detected long periods of stability in population structure and environmental conditions in South Center Lake that were followed by a dramatic temporal shift in population genetic structure after the onset of European settlement and industrialized agriculture in its watershed. In particular, we noted a 24.3-fold increase in phosphorus (P) flux between pre-European and modern sediment P accumulation rates (AR) in this lake. In contrast, no such shifts were detected in Hill Lake, where the watershed was not as impacted by European settlement and rates of change were less directional with a much smaller increase in sediment P AR (2.3-fold). We identify direct and indirect effects of eutrophication proxies on genetic structure in these lake populations and demonstrate the power of using this approach in understanding the consequences of anthropogenic environmental change on natural populations throughout historic time periods.

Environment not dispersal limitation drives clonal composition of Arctic Daphnia in a recently deglaciated area.

One of the most prominent manifestations of the ongoing climate warming is the retreat of glaciers and ice sheets around the world. Retreating glaciers result in the formation of new ponds and lakes, which are available for colonization. The gradual appearance of these new habitat patches allows us to determine to what extent the composition of asexual Daphnia (water flea) populations is affected by environmental drivers vs. dispersal limitation. Here, we used a landscape genetics approach to assess the processes structuring the clonal composition of species in the D. pulex species complex that have colonized periglacial habitats created by ice-sheet retreat in western Greenland. We analysed 61 populations from a young (<50 years) and an old cluster (>150 years) of lakes and ponds. We identified 42 asexual clones that varied widely in spatial distribution. Beta-diversity was higher among older than among younger systems. Lineage sorting by the environment explained 14% of the variation in clonal composition whereas the pure effect of geographical distance was very small and statistically insignificant (Radj2 = 0.010, P = 0.085). Dispersal limitation did not seem important, even among young habitat patches. The observation of several tens of clones colonizing the area combined with environmentally driven clonal composition of populations illustrates that population assembly of asexual species in the Arctic is structured by environmental gradients reflecting differences in the ecology of clones.

Colonization history and clonal richness of asexual Daphnia in periglacial habitats of contrasting age in West Greenland.

Due to climate change, Arctic ice sheets are retreating. This leads to the formation of numerous new periglacial ponds and lakes, which are being colonized by planktonic organisms such as the water flea Daphnia. This system provides unique opportunities to test genotype colonization dynamics and the genetic assemblage of populations. Here, we studied clonal richness of the Daphnia pulex species complex in novel periglacial habitats created by glacial retreat in the Jakobshavn Isbrae area of western Greenland. Along a 10 km transect, we surveyed 73 periglacial habitats out of which 61 were colonized by Daphnia pulex. Hence, for our analysis, we used 21 ponds and 40 lakes in two clusters of habitats differing in age (estimated <50 years vs. >150 years). We tested the expectation that genetic diversity would be low in recently formed (i.e. young), small habitats, but would increase with increasing age and size. We identified a total of 42 genetically distinct clones belonging to two obligately asexual species of the D. pulex species complex: D. middendorffiana and the much more abundant D. pulicaria. While regional clonal richness was high, most clones were rare: 16 clones were restricted to a single habitat and the five most widespread clones accounted for 68% of all individuals sampled. On average, 3·2 clones (range: 1-12) coexisted in a given pond or lake. There was no relationship between clonal richness and habitat size when we controlled for habitat age. Whereas clonal richness was statistically higher in the cluster of older habitats when compared with the cluster of younger ponds and lakes, most young habitats were colonized by multiple genotypes. Our data suggest that newly formed (periglacial) ponds and lakes are colonized within decades by multiple genotypes via multiple colonization events, even in the smallest of our study systems (4 m(2) ).

Characterization of genome-wide SNPs for the water flea Daphnia pulicaria generated by genotyping-by-sequencing (GBS).

The keystone aquatic herbivore Daphnia has been studied for more than 150 years in the context of evolution, ecology and ecotoxicology. Although it is rapidly becoming an emergent model for environmental and population genomics, there have been limited genome-wide level studies in natural populations. We report a unique resource of novel Single Nucleotide Polymorphic (SNP) markers for Daphnia pulicaria using the reduction in genomic complexity with the restriction enzymes approach, genotyping-by-sequencing. Using the genome of D. pulex as a reference, SNPs were scored for 53 clones from five natural populations that varied in lake trophic status. Our analyses resulted in 32,313 highly confident and bi-allelic SNP markers. 1,364 outlier SNPs were mapped on the annotated D. pulex genome, which identified 2,335 genes, including 565 within functional genes. Out of 885 EuKaryotic Orthologous Groups that we found from outlier SNPs, 294 were involved in three metabolic and four regulatory pathways. Bayesian-clustering analyses showed two distinct population clusters representing the possible combined effects of geography and lake trophic status. Our results provide an invaluable tool for future population genomics surveys in Daphnia targeting informative regions related to physiological processes that can be linked to the ecology of this emerging eco-responsive taxon.

Differential transcriptomic responses of ancient and modern Daphnia genotypes to phosphorus supply.

Little is known about the role of transcriptomic changes in driving phenotypic evolution in natural populations, particularly in response to anthropogenic environmental change. Previous analyses of Daphnia genotypes separated by centuries of evolution in a lake using methods in resurrection ecology revealed striking genetic and phenotypic shifts that were highly correlated with anthropogenic environmental change, specifically phosphorus (P)-driven nutrient enrichment (i.e. eutrophication). Here, we compared the transcriptomes of two ancient (~700-year-old) and two modern (~10-year-old) genotypes in historic (low P) and contemporary (high P) environmental conditions using microarrays. We found considerable transcriptomic variation between 'ancient' and 'modern' genotypes in both treatments, with stressful (low P) conditions eliciting differential expression (DE) of a larger number of genes. Further, more genes were DE between 'ancient' and 'modern' genotypes than within these groups. Expression patterns of individual genes differed greatly among genotypes, suggesting that different transcriptomic responses can result in similar phenotypes. While this confounded patterns between 'ancient' and 'modern' genotypes at the gene level, patterns were discernible at the functional level: annotation of DE genes revealed particular enrichment of genes involved in metabolic pathways in response to P-treatments. Analyses of gene families suggested significant DE in pathways already known to be important in dealing with P-limitation in Daphnia as well as in other organisms. Such observations on genotypes of a single natural population, separated by hundreds of years of evolution in contrasting environmental conditions before and during anthropogenic environmental changes, highlight the important role of transcriptional mechanisms in the evolutionary responses of populations.

Functional genomics of intraspecific variation in carbon and phosphorus kinetics in Daphnia.

Understanding how the genome interacts with the environment to produce a diversity of phenotypes is a central challenge in biology. However, we know little about how traits involved in nutrient processing interact with key ecological parameters, such as the supply of mineral nutrients, particularly in animals. The framework of ecological stoichiometry uses information on the content of key elements such as carbon (C) and phosphorus (P) in individuals to predict the success of species. Nevertheless, intraspecific variation in content and the underlying mechanisms that generate such variation has been poorly explored. We studied two genotypes (G1 and G2) of Daphnia pulex that exhibit striking genotype × environment (G × E) interaction in response to shifts in dietary stoichiometry (C:P). G1 had higher fitness under C:P ∼ 100 diet, while G2 performed better in C:P ∼ 800. Dual (14) C/(33) P radiotracer assays show that G1 was more efficient in C processing, while G2 was more efficient in P use. Microarrays revealed that after 3 days of incubation, the genotypes differentially expressed ∼ 25% (7,224) of the total genes on the array under C:P ∼ 100 diet, and ∼ 30% (8,880) of genes under C:P ∼ 800. These results indicate large differences in C and P use between two coexisting genotypes. Importantly, such physiological differences can arise via differential expression of the genome due to alterations in dietary stoichiometry. Basic frameworks such as ecological stoichiometry enable integration of physiological and transcriptomic data, and represent initial steps toward understanding the interplay between fundamental ecological parameters such as nutrient supply and important evolutionary processes such as G × E interactions.

A millennial-scale chronicle of evolutionary responses to cultural eutrophication in Daphnia.

For an accurate assessment of the anthropogenic impacts on evolutionary change in natural populations, we need long-term environmental, genetic and phenotypic data that predate human disturbances. Analysis of c. 1600 years of history chronicled in the sediments of South Center Lake, Minnesota, USA, revealed major environmental changes beginning c. 120 years ago coinciding with the initiation of industrialised agriculture in the catchment area. Population genetic structure, analysed using DNA from dormant eggs of the keystone aquatic herbivore, Daphnia pulicaria, suggested no change for c. 1500 years prior to striking shifts associated with anthropogenic environmental alterations. Furthermore, phenotypic assays on the oldest resurrected metazoan genotypes (potentially as old as c. 700 years) indicate significant shifts in phosphorus utilisation rates compared to younger genotypes. Younger genotypes show steeper reaction norms with high growth under high phosphorus (P), and low growth under low P, while 'ancient' genotypes show flat reaction norms, yet higher growth efficiency under low P. Using this resurrection ecology approach, environmental, genetic and phenotypic data spanning pre- and post-industrialised agricultural eras clearly reveal the evolutionary consequences of anthropogenic environmental change.

The evolutionary time machine: using dormant propagules to forecast how populations can adapt to changing environments.

Evolutionary changes are determined by a complex assortment of ecological, demographic, and adaptive histories. Predicting how evolution will shape the genetic structures of populations coping with current (and future) environmental challenges has principally relied on investigations through space, in lieu of time, because long-term phenotypic and molecular data are scarce. Yet, dormant propagules in sediments, soils, and permafrost are convenient natural archives of population histories from which to trace adaptive trajectories along extended time periods. DNA sequence data obtained from these natural archives, combined with pioneering methods for analyzing both ecological and population genomic time-series data, are likely to provide predictive models to forecast evolutionary responses of natural populations to environmental changes resulting from natural and anthropogenic stressors, including climate change.

The evolution of salinity tolerance in Daphnia: a functional genomics approach.

One route to genetic adaptation in a novel environment is the evolution of ecological generalisation. Yet, identifying the cost that a generalist pays for the increased breadth of tolerance has proven elusive. We integrate phenotypic assays with functional genomics to understand how tolerance to a salinity gradient evolves, and we test the relationship between the fitness cost of this generalisation and the cost of transcription that arises from evolved differences in patterns of gene expression. Our results suggest that a salt-tolerant genotype of Daphnia is characterised by constitutively expressed genes, which does not incur a loss of fitness or a cost of transcription relative to a salt-intolerant genotype in low saline environments. We find that many genes whose expression pattern evolved in response to salinity are also involved in the response to predators, suggesting that the cost of generalisation may be due to trade-offs along other environmental axes.