SAG-RAD: A Method for Single-Cell Population Genomics of Unicellular Eukaryotes.

Sequencing of reduced representation libraries enables genotyping of many individuals for population genomic studies. However, high amounts of DNA are required, and the method cannot be applied directly on single cells, preventing its use on most microbes. We developed and implemented the analysis of single amplified genomes followed by restriction-site-associated DNA sequencing to bypass labor-intensive culturing and to avoid culturing bias in population genomic studies of unicellular eukaryotes. This method thus opens the way for addressing important questions about the genetic diversity, gene flow, adaptation, dispersal, and biogeography of hitherto unexplored species.

Genome-wide single nucleotide polymorphism markers reveal population structure and dispersal direction of an expanding nuisance algal bloom species.

Species invasion and range expansion are currently under scrutiny due to increasing anthropogenic impact on the natural environment. This is also true for harmful algal blooms, which have been reported to have increased in frequency. However, this research is challenging due to the ephemeral nature, small size and mostly low concentrations of microalgae in the environment. One such species is the nuisance microalga Gonyostomum semen (Raphidophyceae), which has increased in occurrence in northern Europe in recent decades. The question of whether the species has expanded its habitat range or if it was already present in the lakes but was too rare to be detected remains unanswered. The aim of the present study was to determine the genetic structure and dispersal pathways of G. semen using RAD (restriction-site-associated DNA) tag sequencing. For G. semen, which has a huge genome (32 Gbp), we faced particular challenges, but were nevertheless able to recover over 1000 single nucleotide polymorphisms at high coverage. Our data revealed a distinct population genetic structure, demonstrating a divide of western and eastern populations that probably represent different lineages. Despite significant genetic differentiation among lakes, we found only limited isolation-by-distance. While we had expected a pattern of recent expansion northwards, the data demonstrated gene flow from the northeast/east towards the southwest/west. This genetic signature suggests that the observed gene flow may be due to dispersal by autumn migratory birds, which act as dispersal vectors of resistant resting propagules that form at the end of the G. semen blooms.

Predation risk and the evolution of a vertebrate stress response: Parallel evolution of stress reactivity and sexual dimorphism.

Predation risk is often invoked to explain variation in stress responses. Yet, the answers to several key questions remain elusive, including the following: (1) how predation risk influences the evolution of stress phenotypes, (2) the relative importance of environmental versus genetic factors in stress reactivity and (3) sexual dimorphism in stress physiology. To address these questions, we explored variation in stress reactivity (ventilation frequency) in a post-Pleistocene radiation of live-bearing fish, where Bahamas mosquitofish (Gambusia hubbsi) inhabit isolated blue holes that differ in predation risk. Individuals of populations coexisting with predators exhibited similar, relatively low stress reactivity as compared to low-predation populations. We suggest that this dampened stress reactivity has evolved to reduce energy expenditure in environments with frequent and intense stressors, such as piscivorous fish. Importantly, the magnitude of stress responses exhibited by fish from high-predation sites in the wild changed very little after two generations of laboratory rearing in the absence of predators. By comparison, low-predation populations exhibited greater among-population variation and larger changes subsequent to laboratory rearing. These low-predation populations appear to have evolved more dampened stress responses in blue holes with lower food availability. Moreover, females showed a lower ventilation frequency, and this sexual dimorphism was stronger in high-predation populations. This may reflect a greater premium placed on energy efficiency in live-bearing females, especially under high-predation risk where females show higher fecundities. Altogether, by demonstrating parallel adaptive divergence in stress reactivity, we highlight how energetic trade-offs may mould the evolution of the vertebrate stress response under varying predation risk and resource availability.

High Iron Requirements for Growth in the Nuisance Alga Gonyostomum semen (Raphidophyceae).

The bloom-forming freshwater alga Gonyostomum semen is associated with acidic, mesotrophic brown water lakes in boreal regions. However, researchers have been unable to conclusively link G. semen abundance and bloom formation to typical brown water lake traits, that is, high water color and DOC (dissolved organic carbon) concentrations. Iron is a main driver of water color in boreal lakes, and a recent study of lake monitoring data indicated a connection between lakes with high G. semen abundance and iron concentrations >200 µg · L-1 . Thus, iron may be the missing link in explaining G. semen abundance and growth dynamics. We experimentally assessed the effects of different iron concentrations above or below 200 µg · L-1 on the growth of G. semen batch monocultures. Iron concentrations <200 µg · L-1 limited G. semen growth, while iron concentrations >200 µg · L-1 did not. Moreover, the iron concentration of the medium required for growth was higher than for other common phytoplankton (Microcystis botrys and Chlamydomonas sp.) included in the experiment. These results indicate that G. semen requires high levels of iron in the lake environment. Consequently, this and previous findings using lake monitoring data support the hypothesis that high concentrations of iron favor the formation of high-density G. semen blooms in boreal brown water lakes. As lakes get browner in a changing climate, monitoring iron levels could be a potential tool to identify lakes at risk for G. semen blooms, especially among lakes that provide ecosystem services to society.

Single-cell genomics of a bloom-forming phytoplankton species reveals population genetic structure across continents.

The study of microbial diversity over time and space is fundamental to the understanding of their ecology and evolution. The underlying processes driving these patterns are not fully resolved but can be studied using population genetic approaches. Here we investigated the population genetic structure of Gonyostomum semen, a bloom-forming phytoplankton species, across two continents. The species appears to be expanding in Europe, whereas similar trends are not observed in the USA. Our aim was to investigate if populations of Gonyostomum semen in Europe and in the USA are genetically differentiated, if there is population genetic structure within the continents, and what the potential drivers of differentiation are. To this end, we used a novel method based on single-amplified genomes combined with Restriction-site Associated DNA sequencing that allows de novo genotyping of natural single-cell isolates without the need for culturing. We amplified over 900 single-cell genomes from 25 lake populations across Europe and the USA and identified two distinct population clusters, one in Europe and another in the USA. Low genetic diversity in European populations supports the hypothesized recent expansion of Gonyostomum semen on this continent. Geographic population structure within each continent was associated with differences in environmental variables that may have led to ecological divergence of population clusters. Overall, our results show that single-amplified genomes combined with Restriction-site Associated DNA sequencing can be used to analyze microalgal population structure and differentiation based on single-cell isolates from natural, uncultured samples.

Low-latitude zooplankton pigmentation plasticity in response to multiple threats.

Crustacean copepods in high-latitude lakes frequently alter their pigmentation facultatively to defend themselves against prevailing threats, such as solar ultraviolet radiation (UVR) and visually oriented predators. Strong seasonality in those environments promotes phenotypic plasticity. To date, no one has investigated whether low-latitude copepods, experiencing continuous stress from UVR and predation threats, exhibit similar inducible defences. We here investigated the pigmentation levels of Bahamian 'blue hole' copepods, addressing this deficit. Examining several populations varying in predation risk, we found the lowest levels of pigmentation in the population experiencing the highest predation pressure. In a laboratory experiment, we found that, in contrast with our predictions, copepods from these relatively constant environments did show some changes in pigmentation subsequent to the removal of UVR; however, exposure to water from different predation regimes induced minor and idiosyncratic pigmentation change. Our findings suggest that low-latitude zooplankton in inland environments may exhibit reduced, but non-zero, levels of phenotypic plasticity compared with their high-latitude counterparts.