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.

Population genomic analyses reveal that salinity and geographic isolation drive diversification in a free-living protist.

Protists make up the vast diversity of eukaryotic life and play a critical role in biogeochemical cycling and in food webs. Because of their small size, cryptic life cycles, and large population sizes, our understanding of speciation in these organisms is very limited. We performed population genomic analyses on 153 strains isolated from eight populations of the recently radiated dinoflagellate genus Apocalathium, to explore the drivers and mechanisms of speciation processes. Species of this genus inhabit both freshwater and saline habitats, lakes and seas, and are found in cold temperate environments across the world. RAD sequencing analyses revealed that the populations were overall highly differentiated, but morphological similarity was not congruent with genetic similarity. While geographic isolation was to some extent coupled to genetic distance, this pattern was not consistent. Instead, we found evidence that the environment, specifically salinity, is a major factor in driving ecological speciation in Apocalathium. While saline populations were unique in loci coupled to genes involved in osmoregulation, freshwater populations appear to lack these. Our study highlights that adaptation to freshwater through loss of osmoregulatory genes may be an important speciation mechanism in free-living aquatic protists.

Rapid diversification of a free-living protist is driven by adaptation to climate and habitat.

Microbial eukaryotes (protists) have major functional roles in aquatic ecosystems, including the biogeochemical cycling of elements as well as occupying various roles in the food web. Despite their importance for ecosystem function, the factors that drive diversification in protists are not known. Here, we aimed to identify the factors that drive differentiation and, subsequently, speciation in a free-living protist, Synura petersenii (Chrysophyceae). We sampled five different geographic areas and utilized population genomics and quantitative trait analyses. Habitat and climate were the major drivers of diversification on the local geographical scale, while geography played a role over longer distances. In addition to conductivity and temperature, precipitation was one of the most important environmental drivers of differentiation. Our results imply that flushing episodes (floods) drive microalgal adaptation to different niches, highlighting the potential for rapid diversification in protists.

Strain-specific metabarcoding reveals rapid evolution of copper tolerance in populations of the coastal diatom Skeletonema marinoi.

Phytoplankton have short generation times, flexible reproduction strategies, large population sizes and high standing genetic diversity, traits that should facilitate rapid evolution under directional selection. We quantified local adaptation of copper tolerance in a population of the diatom Skeletonema marinoi from a mining-exposed inlet in the Baltic Sea and in a non-exposed population 100 km away. We hypothesized that mining pollution has driven evolution of elevated copper tolerance in the impacted population of S. marinoi. Assays of 58 strains originating from sediment resting stages revealed no difference in the average tolerance to copper between the two populations. However, variation within populations was greater at the mining site, with three strains displaying hyper-tolerant phenotypes. In an artificial evolution experiment, we used a novel intraspecific metabarcoding locus to track selection and quantify fitness of all 58 strains during co-cultivation in one control and one toxic copper treatment. As expected, the hyper-tolerant strains enabled rapid evolution of copper tolerance in the mining-exposed population through selection on available strain diversity. Within 42 days, in each experimental replicate a single strain dominated (30%-99% abundance) but different strains dominated the different treatments. The reference population developed tolerance beyond expectations primarily due to slowly developing plastic response in one strain, suggesting that different modes of copper tolerance are present in the two populations. Our findings provide novel empirical evidence that standing genetic diversity of phytoplankton resting stage allows populations to evolve rapidly (20-50 generations) and flexibly on timescales relevant for seasonal bloom progressions.

Single-cell adaptations shape evolutionary transitions to multicellularity in green algae.

The evolution of multicellular life has played a pivotal role in shaping biological diversity. However, we know surprisingly little about the natural environmental conditions that favour the formation of multicellular groups. Here we experimentally examine how key environmental factors (predation, nitrogen and water turbulence) combine to influence multicellular group formation in 35 wild unicellular green algae strains (19 Chlorophyta species). All environmental factors induced the formation of multicellular groups (more than four cells), but there was no evidence this was adaptive, as multicellularity (% cells in groups) was not related to population growth rate under any condition. Instead, population growth was related to extracellular matrix (ECM) around single cells and palmelloid formation, a unicellular life-cycle stage where two to four cells are retained within a mother-cell wall after mitosis. ECM production increased with nitrogen levels resulting in more cells being in palmelloids and higher rates of multicellular group formation. Examining the distribution of 332 algae species across 478 lakes monitored over 55 years, showed that ECM and nitrogen availability also predicted patterns of obligate multicellularity in nature. Our results highlight that adaptations of unicellular organisms to cope with environmental challenges may be key to understanding evolutionary routes to multicellular life.

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.

Local adaptation through countergradient selection in northern populations of Skeletonema marinoi.

Marine microorganisms have the potential to disperse widely with few obvious barriers to gene flow. However, among microalgae, several studies have demonstrated that species can be highly genetically structured with limited gene flow among populations, despite hydrographic connectivity. Ecological differentiation and local adaptation have been suggested as drivers of such population structure. Here we tested whether multiple strains from two genetically distinct Baltic Sea populations of the diatom Skeletonema marinoi showed evidence of local adaptation to their local environments: the estuarine Bothnian Sea and the marine Kattegat Sea. We performed reciprocal transplants of multiple strains between culture media based on water from the respective environments, and we also allowed competition between strains of estuarine and marine origin in both salinities. When grown alone, both marine and estuarine strains performed best in the high-salinity environment, and estuarine strains always grew faster than marine strains. This result suggests local adaptation through countergradient selection, that is, genetic effects counteract environmental effects. However, the higher growth rate of the estuarine strains appears to have a cost in the marine environment and when strains were allowed to compete, marine strains performed better than estuarine strains in the marine environment. Thus, other traits are likely to also affect fitness. We provide evidence that tolerance to pH could be involved and that estuarine strains that are adapted to a more fluctuating pH continue growing at higher pH than marine strains.

Intraspecific variation in metal tolerance modulate competition between two marine diatoms.

Despite widespread metal pollution of coastal ecosystems, little is known of its effect on marine phytoplankton. We designed a co-cultivation experiment to test if toxic dose-response relationships can be used to predict the competitive outcome of two species under metal stress. Specifically, we took into account intraspecific strain variation and selection. We used 72 h dose-response relationships to model how silver (Ag), cadmium (Cd), and copper (Cu) affect both intraspecific strain selection and competition between taxa in two marine diatoms (Skeletonema marinoi and Thalassiosira baltica). The models were validated against 10-day co-culture experiments, using four strains per species. In the control treatment, we could predict the outcome using strain-specific growth rates, suggesting low levels of competitive interactions between the species. Our models correctly predicted which species would gain a competitive advantage under toxic stress. However, the absolute inhibition levels were confounded by the development of chronic toxic stress, resulting in a higher long-term inhibition by Cd and Cu. We failed to detect species differences in average Cu tolerance, but the model accounting for strain selection accurately predicted a competitive advantage for T. baltica. Our findings demonstrate the importance of incorporating multiple strains when determining traits and when performing microbial competition experiments.

Seasonal genotype dynamics of a marine dinoflagellate: Pelagic populations are homogeneous and as diverse as benthic seed banks.

Genetic diversity is the basis for evolutionary adaptation and selection under changing environmental conditions. Phytoplankton populations are genotypically diverse, can become genetically differentiated within small spatiotemporal scales and many species form resting stages. Resting stage accumulations in sediments (seed banks) are expected to serve as reservoirs for genetic information, but so far their role in maintaining phytoplankton diversity and in evolution has remained unclear. In this study we used the toxic dinoflagellate Alexandrium ostenfeldii (Dinophyceae) as a model organism to investigate if (i) the benthic seed bank is more diverse than the pelagic population and (ii) the pelagic population is seasonally differentiated. Resting stages (benthic) and plankton (pelagic) samples were collected at a coastal bloom site in the Baltic Sea, followed by cell isolation and genotyping using microsatellite markers (MS) and restriction site associated DNA sequencing (RAD). High clonal diversity (98%-100%) combined with intermediate to low gene diversity (0.58-0.03, depending on the marker) was found. Surprisingly, the benthic and pelagic fractions of the population were equally diverse, and the pelagic fraction was temporally homogeneous, despite seasonal fluctuation of environmental selection pressures. The results of this study suggest that continuous benthic-pelagic coupling, combined with frequent sexual reproduction, as indicated by persistent linkage equilibrium, prevent the dominance of single clonal lineages in a dynamic environment. Both processes harmonize the pelagic with the benthic population and thus prevent seasonal population differentiation. At the same time, frequent sexual reproduction and benthic-pelagic coupling maintain high clonal diversity in both habitats.

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.

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.

Differences in metal tolerance among strains, populations, and species of marine diatoms - Importance of exponential growth for quantification.

Strains of microalgae vary in traits between species and populations due to adaptation or stochastic processes. Traits of individual strains may also vary depending on the acclimatization state and external forces, such as abiotic stress. In this study we tested how metal tolerance differs among marine diatoms at three organizational levels: species, populations, and strains. At the species level we compared two pelagic Baltic Sea diatoms (Skeletonema marinoi and Thalassiosira baltica). We found that the between-species differences in tolerance (EC50) to the biologically active metals (Cu, Co, Ni, and Zn) was similar to that within-species. In contrast, the two species differed significantly in tolerance towards the non-essential metals, Ag (three-fold higher in T. baltica), Pb and Cd (two and three-fold higher in S. marinoi). At the population level, we found evidence that increased tolerance against Cu and Co (17 and 41 % higher EC50 on average, respectively) had evolved in a S. marinoi population subjected to historical mining activity. On a strain level we demonstrate how the growth phase of cultures (i.e., cellular densities above exponential growth) modulated dose-response relationships to Ag, Cd, Co, Cu, and Zn. Specifically, the EC50's were reduced by 10-60 % in non-exponentially growing S. marinoi (strain RO5AC), depending on metal. For the essential metals these differences were often larger than the average differences between the two species and populations. Consequently, without careful experimental design, interactions between nutrient limitation and metal stress may interfere with detection of small, but evolutionary and ecologically important, differences in tolerance between microalgae. To avoid such artifacts, we outline a semi-continuous cultivation approach that maintains, and empirically tests, that exponential growth is achieved. We argue that such an approach is essential to enable comparison of population or strain differences in tolerance using dose-response tests on cultures of microalgae.

High Diversity of Microcystin Chemotypes within a Summer Bloom of the Cyanobacterium Microcystis botrys.

The fresh-water cyanobacterium Microcystis is known to form blooms world-wide, and is often responsible for the production of microcystins found in lake water. Microcystins are non-ribosomal peptides with toxic effects, e.g. on vertebrates, but their function remains largely unresolved. Moreover, not all strains produce microcystins, and many different microcystin variants have been described. Here we explored the diversity of microcystin variants within Microcystis botrys, a common bloom-former in Sweden. We isolated a total of 130 strains through the duration of a bloom in eutrophic Lake Vomb, and analyzed their microcystin profiles with tandem mass spectrometry (LC-MS/MS). We found that microcystin producing (28.5%) and non-producing (71.5%) M. botrys strains, co-existed throughout the bloom. However, microcystin producing strains were more prevalent towards the end of the sampling period. Overall, 26 unique M. botrys chemotypes were identified, and while some chemotypes re-occurred, others were found only once. The M. botrys chemotypes showed considerable variation both in terms of number of microcystin variants, as well as in what combinations the variants occurred. To our knowledge, this is the first report on microcystin chemotype variation and dynamics in M. botrys. In addition, our study verifies the co-existence of microcystin and non-microcystin producing strains, and we propose that environmental conditions may be implicated in determining their composition.

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.

Sequencing and Phylogenetic Analysis of Chloroplast Genes in Freshwater Raphidophytes.

The complex evolution of chloroplasts in microalgae has resulted in highly diverse pigment profiles. Freshwater raphidophytes, for example, display a very different pigment composition to marine raphidophytes. To investigate potential differences in the evolutionary origin of chloroplasts in these two groups of raphidophytes, the plastid genomes of the freshwater species Gonyostomumsemen and Vacuolariavirescens were sequenced. To exclusively sequence the organelle genomes, chloroplasts were manually isolated and amplified using single-cell whole-genome-amplification. Assembled and annotated chloroplast genes of the two species were phylogenetically compared to the marine raphidophyte Heterosigmaakashiwo and other evolutionarily more diverse microalgae. These phylogenetic comparisons confirmed the high relatedness of all investigated raphidophyte species despite their large differences in pigment composition. Notable differences regarding the presence of light-independent protochlorophyllide oxidoreductase (LIPOR) genes among raphidophyte algae were also revealed in this study. The whole-genome amplification approach proved to be useful for isolation of chloroplast DNA from nuclear DNA. Although only approximately 50% of the genomes were covered, this was sufficient for a multiple gene phylogeny representing large parts of the chloroplast genes.

The potential for dispersal of microalgal resting cysts by migratory birds.

Most microalgal species are geographically widespread, but little is known about how they are dispersed. One potential mechanism for long-distance dispersal is through birds, which may transport cells internally (endozoochory) and deposit them during, or in-between, their migratory stopovers. We hypothesize that dinoflagellates, in particular resting stages, can tolerate bird digestion; that bird temperature, acidity, and retention time negatively affect dinoflagellate viability; and that recovered cysts can germinate after passage through the birds' gut, contributing to species-specific dispersal of the dinoflagellates across scales. Tolerance of two dinoflagellate species (Peridiniopsis borgei, a warm-water species and Apocalathium malmogiense, a cold-water species) to Mallard gut passage was investigated using in vitro experiments simulating the gizzard and caeca conditions. The effect of in vitro digestion and retention time on cell integrity, cell viability, and germination capacity of the dinoflagellate species was examined targeting both their vegetative and resting stages. Resting stages (cysts) of both species were able to survive simulated bird gut passage, even if their survival rate and germination were negatively affected by exposure to acidic condition and bird internal temperature. Cysts of A. malmogiense were more sensitive than P. borgei to treatments and to the presence of digestive enzymes. Vegetative cells did not survive conditions of bird internal temperature and formed pellicle cysts when exposed to gizzard-like acid conditions. We show that dinoflagellate resting cysts serve as dispersal propagules through migratory birds. Assuming a retention time of viable cysts of 2-12 h to duck stomach conditions, cysts could be dispersed 150-800 km and beyond.

Contrasting prevalence of selection and drift in the community structuring of bacteria and microbial eukaryotes.

Whether or not communities of microbial eukaryotes are structured in the same way as bacteria is a general and poorly explored question in ecology. Here, we investigated this question in a set of planktonic lake microbiotas in Eastern Antarctica that represent a natural community ecology experiment. Most of the analysed lakes emerged from the sea during the last 6000 years, giving rise to waterbodies that originally contained marine microbiotas and that subsequently evolved into habitats ranging from freshwater to hypersaline. We show that habitat diversification has promoted selection driven by the salinity gradient in bacterial communities (explaining ∼ 72% of taxa turnover), while microeukaryotic counterparts were predominantly structured by ecological drift (∼72% of the turnover). Nevertheless, we also detected a number of microeukaryotes with specific responses to salinity, indicating that albeit minor, selection has had a role in the structuring of specific members of their communities. In sum, we conclude that microeukaryotes and bacteria inhabiting the same communities can be structured predominantly by different processes. This should be considered in future studies aiming to understand the mechanisms that shape microbial assemblages.

A planktonic diatom displays genetic structure over small spatial scales.

Marine planktonic microalgae have potentially global dispersal, yet reduced gene flow has been confirmed repeatedly for several species. Over larger distances (>200 km) geographic isolation and restricted oceanographic connectivity have been recognized as instrumental in driving population divergence. Here we investigated whether similar patterns, that is, structured populations governed by geographic isolation and/or oceanographic connectivity, can be observed at smaller (6-152 km) geographic scales. To test this we established 425 clonal cultures of the planktonic diatom Skeletonema marinoi collected from 11 locations in the Archipelago Sea (northern Baltic Sea). The region is characterized by a complex topography, entailing several mixing regions of which four were included in the sampling area. Using eight microsatellite markers and conventional F-statistics, significant genetic differentiation was observed between several sites. Moreover, Bayesian cluster analysis revealed the co-occurrence of two genetic groups spread throughout the area. However, geographic isolation and oceanographic connectivity could not explain the genetic patterns observed. Our data reveal hierarchical genetic structuring whereby despite high dispersal potential, significantly diverged populations have developed over small spatial scales. Our results suggest that biological characteristics and historical events may be more important in generating barriers to gene flow than physical barriers at small spatial scales.

Delineating closely related dinoflagellate lineages using phylotranscriptomics.

Recently radiated dinoflagellates Apocalathium aciculiferum (collected in Lake Erken, Sweden), Apocalathium malmogiense (Baltic Sea) and Apocalathium aff. malmogiense (Highway Lake, Antarctica) represent a lineage with an unresolved phylogeny. We determined their phylogenetic relationships using phylotranscriptomics based on 792 amino acid sequences. Our results showed that A. aciculiferum diverged from the other two closely related lineages, consistent with their different morphologies in cell size, relative cell length and presence of spines. We hypothesized that A. aff. malmogiense and A. malmogiense, which inhabit different hemispheres, are evolutionarily more closely related because they diverged from a marine common ancestor, adapting to a wide salinity range, while A. aciculiferum colonized a freshwater habitat, by acquiring adaptations to this environment, in particular, salinity intolerance. We show that phylotranscriptomics can resolve the phylogeny of recently diverged protists. This has broad relevance, given that many phytoplankton species are morphologically very similar, and single genes sometimes lack the information to determine species' relationships.

Spatio-Temporal Interdependence of Bacteria and Phytoplankton during a Baltic Sea Spring Bloom.

In temperate systems, phytoplankton spring blooms deplete inorganic nutrients and are major sources of organic matter for the microbial loop. In response to phytoplankton exudates and environmental factors, heterotrophic microbial communities are highly dynamic and change their abundance and composition both on spatial and temporal scales. Yet, most of our understanding about these processes comes from laboratory model organism studies, mesocosm experiments or single temporal transects. Spatial-temporal studies examining interactions of phytoplankton blooms and bacterioplankton community composition and function, though being highly informative, are scarce. In this study, pelagic microbial community dynamics (bacteria and phytoplankton) and environmental variables were monitored during a spring bloom across the Baltic Proper (two cruises between North Germany to Gulf of Finland). To test to what extent bacterioplankton community composition relates to the spring bloom, we used next generation amplicon sequencing of the 16S rRNA gene, phytoplankton diversity analysis based on microscopy counts and population genotyping of the dominating diatom Skeletonema marinoi. Several phytoplankton bloom related and environmental variables were identified to influence bacterial community composition. Members of Bacteroidetes and Alphaproteobacteria dominated the bacterial community composition but the bacterial groups showed no apparent correlation with direct bloom related variables. The less abundant bacterial phyla Actinobacteria, Planctomycetes, and Verrucomicrobia, on the other hand, were strongly associated with phytoplankton biomass, diatom:dinoflagellate ratio, and colored dissolved organic matter (cDOM). Many bacterial operational taxonomic units (OTUs) showed high niche specificities. For example, particular Bacteroidetes OTUs were associated with two distinct genetic clusters of S. marinoi. Our study revealed the complexity of interactions of bacterial taxa with inter- and intraspecific genetic variation in phytoplankton. Overall, our findings imply that biotic and abiotic factors during spring bloom influence bacterial community dynamics in a hierarchical manner.