Understanding stoichiometric constraints on growth using resource use efficiency imbalances.

Growth is a function of the net accrual of resources by an organism. Energy and elemental contents of organisms are dynamically linked through their uptake and allocation to biomass production, yet we lack a full understanding of how these dynamics regulate growth rate. Here, we develop a multivariate imbalance framework, the growth efficiency hypothesis, linking organismal resource contents to growth and metabolic use efficiencies, and demonstrate its effectiveness in predicting consumer growth rates under elemental and food quantity limitation. The relative proportions of carbon (%C), nitrogen (%N), phosphorus (%P), and adenosine triphosphate (%ATP) in consumers differed markedly across resource limitation treatments. Differences in their resource composition were linked to systematic changes in stoichiometric use efficiencies, which served to maintain relatively consistent relationships between elemental and ATP content in consumer tissues and optimize biomass production. Overall, these adjustments were quantitatively linked to growth, enabling highly accurate predictions of consumer growth rates.

SEED: A framework for integrating ecological stoichiometry and eco-evolutionary dynamics.

Characterising the extent and sources of intraspecific variation and their ecological consequences is a central challenge in the study of eco-evolutionary dynamics. Ecological stoichiometry, which uses elemental variation of organisms and their environment to understand ecosystem patterns and processes, can be a powerful framework for characterising eco-evolutionary dynamics. However, the current emphasis on the relative content of elements in the body (i.e. organismal stoichiometry) has constrained its application. Intraspecific variation in the rates at which elements are acquired, assimilated, allocated or lost is often greater than the variation in organismal stoichiometry. There is much to gain from studying these traits together as components of an 'elemental phenotype'. Furthermore, each of these traits can have distinct ecological effects that are underappreciated in the current literature. We propose a conceptual framework that explores how microevolutionary change in the elemental phenotype occurs, how its components interact with each other and with other traits, and how its changes can affect a wide range of ecological processes. We demonstrate how the framework can be used to generate novel hypotheses and outline pathways for future research that enhance our ability to explain, analyse and predict eco-evolutionary dynamics.

Adaptation to a limiting element involves mitigation of multiple elemental imbalances.

About 20 elements underlie biology and thus constrain biomass production. Recent systems-level observations indicate that altered supply of one element impacts the processing of most elements encompassing an organism (i.e. ionome). Little is known about the evolutionary tendencies of ionomes as populations adapt to distinct biogeochemical environments. We evolved the bacterium Serratia marcescens under five conditions (i.e. low carbon, nitrogen, phosphorus, iron or manganese) that limited the yield of the ancestor compared with replete medium, and measured the concentrations and use efficiency of these five, and five other elements. Both physiological responses of the ancestor, as well as evolutionary responses of descendants to experimental environments involved changes in the content and use efficiencies of the limiting element, and several others. Differences in coefficients of variation in elemental contents based on biological functions were evident, with those involved in biochemical building (C, N, P, S) varying least, followed by biochemical balance (Ca, K, Mg, Na), and biochemical catalysis (Fe, Mn). Finally, descendants evolved to mitigate elemental imbalances evident in the ancestor in response to limiting conditions. Understanding the tendencies of such ionomic responses will be useful to better forecast biological responses to geochemical changes.

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.

Revisiting the growth rate hypothesis: Towards a holistic stoichiometric understanding of growth.

The growth rate hypothesis (GRH) posits that variation in organismal stoichiometry (C:P and N:P ratios) is driven by growth-dependent allocation of P to ribosomal RNA. The GRH has found broad but not uniform support in studies across diverse biota and habitats. We synthesise information on how and why the tripartite growth-RNA-P relationship predicted by the GRH may be uncoupled and outline paths for both theoretical and empirical work needed to broaden the working domain of the GRH. We found strong support for growth to RNA (r2  = 0.59) and RNA-P to P (r2  = 0.63) relationships across taxa, but growth to P relationships were relatively weaker (r2  = 0.09). Together, the GRH was supported in ~50% of studies. Mechanisms behind GRH uncoupling were diverse but could generally be attributed to physiological (P accumulation in non-RNA pools, inactive ribosomes, translation elongation rates and protein turnover rates), ecological (limitation by resources other than P), and evolutionary (adaptation to different nutrient supply regimes) causes. These factors should be accounted for in empirical tests of the GRH and formalised mathematically to facilitate a predictive understanding of growth.

Impacts of heavy metal pollution on the ionomes and transcriptomes of Western mosquitofish (Gambusia affinis).

Our understanding of the mechanisms mediating the resilience of organisms to environmental change remains lacking. Heavy metals negatively affect processes at all biological scales, yet organisms inhabiting contaminated environments must maintain homeostasis to survive. Tar Creek in Oklahoma, USA, contains high concentrations of heavy metals and an abundance of Western mosquitofish (Gambusia affinis), though several fish species persist at lower frequency. To test hypotheses about the mechanisms mediating the persistence and abundance of mosquitofish in Tar Creek, we integrated ionomic data from seven resident fish species and transcriptomic data from mosquitofish. We predicted that mosquitofish minimize uptake of heavy metals more than other Tar Creek fish inhabitants and induce transcriptional responses to detoxify metals that enter the body, allowing them to persist in Tar Creek at higher density than species that may lack these responses. Tar Creek populations of all seven fish species accumulated heavy metals, suggesting mosquitofish cannot block uptake more efficiently than other species. We found population-level gene expression changes between mosquitofish in Tar Creek and nearby unpolluted sites. Gene expression differences primarily occurred in the gill, where we found upregulation of genes involved with lowering transfer of metal ions from the blood into cells and mitigating free radicals. However, many differentially expressed genes were not in known metal response pathways, suggesting multifarious selective regimes and/or previously undocumented pathways could impact tolerance in mosquitofish. Our systems-level study identified well characterized and putatively new mechanisms that enable mosquitofish to inhabit heavy metal-contaminated environments.

Growth and ionomic responses of a freshwater cyanobacterium to supplies of nitrogen and iron.

Cyanobacterial harmful algal blooms (HABs) are increasing in frequency and magnitude worldwide. A number of parameters are thought to underlie HABs, including the ratio at which two key elements, nitrogen (N) and phosphorus (P) are supplied, although a predictive understanding eludes us. While the physiological importance of iron (Fe) in electron transport and N-fixation is well known, relatively little is known about its impacts on the growth of freshwater cyanobacteria. Moreover, there is growing appreciation for correlated changes in the quotas of multiple elements encompassing an organism (i.e. the ionome) when the supply of one element changes, indicating that growth differences arise from complex biochemical adjustments rather than limitation of a key anabolic process by a single element. In this study, the effects of supply N:P and Fe on the growth and ionome of Dolichospermum, a nitrogen-fixing cyanobacterium found in freshwater ecosystems, were examined. Changes in both supply N:P and Fe had significant effects on yield. Consistent with prior observations, cyanobacterial growth was higher at N:P = 20, compared to N:P = 5, and quotas of all elements decreased with growth. Yield was negatively related with the degree of imbalance between dissolved supply and intracellular concentrations of not only N and Fe, but also multiple other elements. Changes in Fe supply had a significant effect on yield in N-limited conditions (N:P = 5). Again, ionome-wide imbalances decreased yield. Together, these results indicate that attention to multiple elements encompassing the ionome of a HAB-forming taxon, and the supplies of such elements may help improve the ability to forecast blooms. Such elemental interactions may be critical as limnologists begin to appreciate the staggering variation in the supplies of such elements among lakes, and anthropogenic activities continue to alter global biogeochemical cycles.

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.

Differential responses of macroinvertebrate ionomes across experimental N:P gradients in detritus-based headwater streams.

Diverse global change processes are reshaping the biogeochemistry of stream ecosystems. Nutrient enrichment is a common stressor that can modify flows of biologically important elements such as carbon (C), nitrogen (N), and phosphorus (P) through stream foodwebs by altering the stoichiometric composition of stream organisms. However, enrichment effects on concentrations of other important essential and trace elements in stream taxa are less understood. We investigated shifts in macroinvertebrate ionomes in response to changes in coarse benthic organic matter (CBOM) stoichiometry following N and P enrichment of five detritus-based headwater streams. Concentrations of most elements (17/19) differed among three insect genera (Maccaffertium sp., Pycnopsyche spp., and Tallaperla spp.) prior to enrichment. Genus-specific changes in the body content of: P, magnesium, and sodium (Na) in Tallaperla; P, Na, and cadmium in Pycnopsyche; and P in Maccaffertium were also found across CBOM N:P gradients. These elements increased in Tallaperla but decreased in the other two taxa due to growth dilution at larger body sizes. Multivariate elemental differences were found across all taxa, and ionome-wide shifts with dietary N and P enrichment were also observed in Tallaperla and Pycnopsyche. Our results show that macroinvertebrates exhibit distinct differences in elemental composition beyond C, N, and P and that the ionomic composition of common stream taxa can vary with body size and N and P enrichment. Thus, bottom-up changes in N and P supplies could potentially influence the cycling of lesser studied biologically essential elements in aquatic environments by altering their relative proportions in animal tissues.

Phosphorus supply shifts the quotas of multiple elements in algae and Daphnia: ionomic basis of stoichiometric constraints.

The growth rate hypothesis posits that the rate of protein synthesis is constrained by phosphorus (P) supply. P scarcity invokes differential expression of genes involved in processing of most if not all elements encompassing an individual (the ionome). Whether such ionome-wide adjustments to P supply impact growth and trophic interactions remains unclear. We quantified the ionomes of a resource-consumer pair in contrasting P supply conditions. Consumer growth penalty was driven by not only P imbalance between trophic levels but also imbalances in other elements, reflecting complex physiological adjustments made by both the resource and the consumer. Mitigating such imbalances requires energy and should impact the efficiency at which assimilated nutrients are converted to biomass. Correlated shifts in the handling of multiple elements, and variation in the supplies of such elements could underlie vast heterogeneity in the rates at which organisms and ecosystems accrue biomass as a function of P supply.

Does differential iron supply to algae affect Daphnia life history? An ionome-wide study.

The availability of iron (Fe) varies considerably among diet items, as well as ecosystems. Availability of Fe has also changed due to anthropogenic environmental changes in oceanic as well as inland ecosystems. We know little about its role in the nutrition of ecologically important consumers, particularly in inland ecosystems. Physiological studies in several taxa indicate marked effects of dietary Fe on oogenesis. We predicted that differential Fe supply to algae will impact algal Fe concentration with consequences on the life history of the freshwater grazer, Daphnia magna. We found that algal Fe concentration increased with Fe supply, but did not affect algal growth, indicating that the majority of experimental Fe additions were likely adsorbed to, or stored in algal cells. Regardless, data indicate that algal Fe impacted the reproductive traits (age and size at maturity) but not juvenile growth rate of Daphnia. A subsequent experiment revealed that Fe concentration in eggs was significantly higher than the rest of Daphnia. These results indicate that the concentration of Fe in or on algal cells may vary considerably among ecosystems overlying distinct geological formations differing in Fe, possibly with important implications for zooplankton life histories. Understanding the mechanisms underlying this response is unlikely to be accomplished by a strict focus on Fe because we found correlated shifts in the algal ionome, with concomitant ionome-wide adjustments in Daphnia. Information on ionome-wide responses may be useful in better understanding the responses of biota to changes in the supply of any one element.

Ionome and elemental transport kinetics shaped by parallel evolution in threespine stickleback.

Evidence that organisms evolve rapidly enough to alter ecological dynamics necessitates investigation of the reciprocal links between ecology and evolution. Data that link genotype to phenotype to ecology are needed to understand both the process and ecological consequences of rapid evolution. Here, we quantified the suite of elements in individuals (i.e., ionome) and differences in the fluxes of key nutrients across populations of threespine stickleback. We find that allelic variation associated with freshwater adaptation that controls bony plating is associated with changes in the ionome and nutrient recycling. More broadly, we find that adaptation of marine stickleback to freshwater conditions shifts the ionomes of natural populations and populations raised in common gardens. In both cases ionomic divergence between populations was primarily driven by differences in trace elements rather than elements typically associated with bone. These findings demonstrate the utility of ecological stoichiometry and the importance of ionome-wide data in understanding eco-evolutionary dynamics.

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.

Assessment of biochemical mechanisms of tolerance to chlorpyrifos in ancient and contemporary Daphnia pulicaria genotypes.

The evolution of tolerance to environmental contaminants in non-target taxa has been largely studied by comparing extant populations experiencing contrasting exposure. Previous research has demonstrated that "resurrected" genotypes from a population of Daphnia pulicaria express temporal variation in sensitivity to the insecticide chlorpyrifos. Ancient genotypes (1301-1646AD.) were on average more sensitive to this chemical compared to the contemporary genotypes (1967-1977AD.). To determine the physiological mechanisms of tolerance, a series of biochemical assays was performed on three ancient and three contemporary genotypes; these six genotypes exhibited the most sensitive and most tolerant phenotypes within the population, respectively. Metabolic tolerance mechanisms were evaluated using acute toxicity testing, while target-site tolerance was assessed via in vitro acetylcholinesterase (AChE) assays. Acute toxicity tests were conducted using i) the toxic metabolite chlorpyrifos-oxon (CPF-oxon) and ii) CPF-oxon co-applied with piperonyl butoxide (PBO), a known Phase-I metabolic inhibitor. Both series of toxicity tests reduced the mean variation in sensitivity between tolerant and sensitive genotypes. Exposure to CPF-O reduced the disparity from a 4.7-fold to 1.6-fold difference in sensitivity. The addition of PBO further reduced the variation to a 1.2-fold difference in sensitivity. In vitro acetylcholinesterase assays yielded no significant differences in constitutive activity or target-site sensitivity. These findings suggest that pathways involving Phase-I detoxification and/or bioactivation of chlorpyrifos play a significant role in dictating the microevolutionary trajectories of tolerance in this population.

Ecological Stoichiometry beyond Redfield: An Ionomic Perspective on Elemental Homeostasis.

Elemental homeostasis has been largely characterized using three important elements that were part of the Redfield ratio (i.e., carbon: nitrogen: phosphorus). These efforts have revealed substantial diversity in homeostasis among taxonomic groups and even within populations. Understanding the evolutionary basis, and ecological consequences of such diversity is a central challenge. Here, we propose that a more complete understanding of homeostasis necessitates the consideration of other elements beyond C, N, and P. Specifically, we posit that physiological complexity underlying maintenance of elemental homeostasis along a single elemental axis impacts processing of other elements, thus altering elemental homeostasis along other axes. Indeed, transcriptomic studies in a wide variety of organisms have found that individuals differentially express significant proportions of the genome in response to variability in supply stoichiometry in order to maintain varying levels of homeostasis. We review the literature from the emergent field of ionomics that has established the consequences of such physiological trade-offs on the content of the entire suite of elements in an individual. Further, we present experimental data on bacteria exhibiting divergent phosphorus homeostasis phenotypes demonstrating the fundamental interconnectedness among elemental quotas. These observations suggest that physiological adjustments can lead to unexpected patterns in biomass stoichiometry, such as correlated changes among suites of non-limiting microelements in response to limitation by macroelements. Including the entire suite of elements that comprise biomass will foster improved quantitative understanding of the links between chemical cycles and the physiology of organisms.

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.