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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.

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.

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.

Evolutionary changes in populations of microbes, such as microalgae, cannot be traced using conventional metabarcoding loci as they lack intraspecific resolution. Consequently, selection and competition processes amongst strains of the same species cannot be resolved without elaborate isolation, culturing, and genotyping efforts. Bamboozle, a new bioinformatic tool introduced here, scans a species’ entire genome and identifies allele-rich barcodes that enable direct identification of different strains from a common population, and a single DNA sample, using amplicon sequencing. We demonstrate its usefulness by identifying hypervariable barcoding loci (Skeletonema marinoi, and the haploid chlorophyteChlamydomonas reinhardtii. Across the genomes, only 26 loci capable of resolving all available strains’ genotypes were identified, all of which are within protein-coding genes of variable metabolic function. Single nucleotide polymorphisms (SNPs) provided the most reliable genetic markers, and amongst 55 strains ofS. marinoi,three 500 bp loci contained, on average, 46 SNPs, 103 unique alleles, and displayed 100% heterozygosity. The prevalence of heterozygosity was identified as a novel opportunity to improve strain quantification and detect false positive artefacts during denoising of amplicon sequences. Finally, we illustrate how metabarcoding of a single genetic locus can be used to track strain abundances of 58 strains ofS. marinoiin an artificial selection experiment. As future genomics datasets become available and DNA sequencing technologies develop, Bamboozle has flexible user settings enabling optimal barcodes to be designed for other species and applications.

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.

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.

A study on the freshwater dinoflagellate (Dinophyceae) of Colombia was carried out based on samples from four swamps, nine reservoirs, a small insular lake, and additional specimens deposited in the phycological collection of Universidad de Antioquia, Colombia, and the collection of Universidad de La Plata, Argentina. For most species, detailed morphological descriptions and illustrations are provided. Moreover, the geographical distribution of the species is updated. Durinskia spp., Glochidinium penardiforme, Naiadinium polonicum, Peridiniopsis elpatiewskyi, Peridiniopsis quadridens, and Sphaerodinium fimbriatum, are reported for the first time in Colombia. Furthermore, the correct sulcal plate pattern was determined for Naidinium polonicum which has been misinterpreted in earlier literature.

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.

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 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.

Predation is a well-studied driver of ecological selection on prey traits, which frequently drives divergence in anti-predator performance across environments that vary in predation risk. However, predation also alters prey mortality regimes, where low predation risk often results in higher prey densities and consequently higher intensities of intraspecific resource competition. In addition, predation risk alters the foraging context, as acquiring food can be risky in the presence of predators. Thus, different predation regimes can drive divergent selection on traits associated with resource competition, such as foraging behaviours. Moreover, because sexes often differ in susceptibility to predation and limitations to their reproductive output, the intensity of the tradeoff between predator avoidance and resource competition may depend on sex. We used a laboratory experiment to assess key aspects of foraging performance in a predator-free context in Bahamas mosquitofish Gambusia hubbsi wild-caught from multiple populations that experience either high or low levels of predation risk. When competing for limited food resources at a common density, females from low-predation regimes showed higher foraging and food consumption rates than females from high-predation regimes. Males showed fewer differences between predation regimes, and an opposite pattern from females. We suggest these sex-specific effects result from females facing a greater tradeoff between predation risk and resource competition, combined with males from high-predation environments elevating foraging behaviours in the absence of nearby predators and females. Females of this species are larger than males, bear live young and show higher foraging rates in the wild than males. On the other hand, males spend more time pursuing females in the wild, and may exhibit greater flexibility in foraging behaviours based on the immediate context. Our results show that varying levels of predation risk can lead to differences in behaviours associated with resource competition, but these effects can strongly differ between sexes. CC BY 3.0First published: 16 March 2021

Diel vertical migration (DVM) is the most common behavioral phenomenon in zooplankton, and numerous studies have evaluated DVM under strong seasonality at higher latitudes. Yet, our understanding of the environmental drivers of DVM at low latitudes, where seasonal variation is less pronounced, remains limited. Therefore, we here examined patterns of vertical distribution in copepods in six subtropical Bahamian blue holes with different food web structure and tested the role of several key environmental variables potentially affecting this behavior. Day and night samplings showed that copepods generally performed DVM, characterized by downward migration to deeper depths during the day and upward migration to surface waters at night. Across all blue holes, the daytime vertical depth distribution of calanoid copepods correlated positively with both predation risk and depth of food resources (Chlorophylla), but was less affected by ultraviolet radiation (UVR). A potential explanation is that since UVR is a continuous threat across seasons, zooplankton have established photoprotective pigmentation making them less vulnerable to this threat. The copepods also showed a size-structured depth segregation, where larger individuals were found at deeper depths during the day, which further strengthens the suggestion that predation is a major driver of DVM in these systems. Hence, in contrast to studies performed at higher latitudes, we show that despite the constant exposure to UVR, predator avoidance and food availability are the most pronounced drivers of copepod DVM at those low latitudes, suggesting that the main driver of DVM may vary among systems, but also systematically by latitude.

Inland waters receive and process large amounts of colored organic matter from the terrestrial surroundings. These inputs dramatically affect the chemical, physical, and biological properties of water bodies, as well as their roles as global carbon sinks and sources. However, manipulative studies, especially at ecosystem scale, require large amounts of dissolved organic matter with optical and chemical properties resembling indigenous organic matter. Here, we compared the impacts of two leonardite products (HuminFeed and SuperHume) and a freshly derived reverse osmosis concentrate of organic matter in a set of comprehensive mesocosm‐ and laboratory‐scale experiments and analyses. The chemical properties of the reverse osmosis concentrate and the leonardite products were very different, with leonardite products being low and the reverse osmosis concentrate being high in carboxylic functional groups. Light had a strong impact on the properties of leonardite products, including loss of color and increased particle formation. HuminFeed presented a substantial impact on microbial communities under light conditions, where bacterial production was stimulated and community composition modified, while in dark potential inhibition of bacterial processes was detected. While none of the browning agents inhibited the growth of the tested phytoplankton Gonyostomum semen, HuminFeed had detrimental effects on zooplankton abundance and Daphnia reproduction. We conclude that the effects of browning agents extracted from leonardite, particularly HuminFeed, are in sharp contrast to those originating from terrestrially derived dissolved organic matter. Hence, they should be used with great caution in experimental studies on the consequences of terrestrial carbon for aquatic systems., Marie Curie International Outgoing Fellowship, Swedish Research Council Formas http://dx.doi.org/10.13039/501100001862, Knut and Alice Wallenberg Foundation http://dx.doi.org/10.13039/501100004063

Microalgal range expansions are increasing in frequency and magnitude but generally remain unnoticed until mass development occurs. Gonyostomum semen is a freshwater raphidophyte that causes nuisance blooms in lakes and has recently expanded its distribution across Europe. G. semen was considered to mainly occur in humic lakes in the boreal region but is now found in high density also in other freshwater habitats on a larger geographic scale with growing incidence. In this study, we focused on which environmental factors limit its expansion. Our hypothesis was that G. semen occurs in many different lake types, except for high alkalinity lakes, in which high pH in combination with high calcium concentration would inhibit its growth. Results from our field study illustrate the environmental heterogeneity of G. semen bloom sites across Europe and the United States. Nevertheless, none of these sites combined high pH and high calcium concentration. In a mesocosm study, as well as a laboratory experiment, we further demonstrated that growth of G. semen is inhibited in conditions combining both high pH and high calcium concentration. We also discuss the function of Sphagnum peat mosses in rendering an alkaline habitat suitable to G. semen growth. Our study highlights that high alkalinity environments act as a major colonization barrier to G. semen. While this finding explains which environmental filters limit G. semen distribution it also helps in understanding its current expansion. With globally decreasing calcium concentrations in freshwater ecosystems, new habitats have and will become conducive to G. semen growth.

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.

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.

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.

Eukaryotic phytoplankton exhibit an enormous species richness, displaying a range of phylogenetic, morphological and physiological diversity. Yet, until recently, very little was known about the diversity, genetic variation and evolutionary processes within species and populations. An approach to explore this diversity and to understand evolution of phytoplankton is to use population genetics as a conceptual framework and methodology. Here, we discuss the patterns, processes and questions that population genetic studies have revealed in eukaryotic phytoplankton. First, we describe the main biological processes generating genetic variation. We specifically discuss the importance of life-cycle complexity for genetic and phenotypic diversity and consider how such diversity can be maintained during blooms when rapid asexual proliferation dominates. Next, we explore how genetic diversity is partitioned over time and space, with a focus on the processes shaping this structure, in particular selection and genetic exchange. Our aim is also to show how population genetics can be used to make inferences about realized dispersal and sexual recombination, as these processes are so difficult to study directly. Finally, we highlight important open questions and suggest promising avenues for future studies that will be made possible by new sequencing technologies

A number of marine and freshwater harmful algal bloom (HAB) species have colonized new areas and expanded their habitat range in recent years. Nevertheless it is notoriously difficult to establish when colonization first occurred, what the dispersal routes are, and to separate recent invasion from increases in existent but small populations. The freshwater raphidophyte Gonyostomum semen is a nuisance species that has expanded its habitat range and increased in abundance in northern Europe during the past decades. To evaluate to what extent sediments can be used for determining historic occurrence of G. semen, a quantitative real-time PCR method for detecting cysts of this algae was developed. This paper presents a qPCR protocol with a set of primers that are specific to Gonyostomum and with PCR conditions optimized for sediment samples from humic lakes, which are the common habitat of G. semen. With this sensitive method as few as 1.6 cysts per PCR reaction could be reliably quantified, corresponding to 320 cysts per g wet weight sediment. Cysts were present in sediments with ages ranging from years to decades and their persistence allows detection of historic populations up to at least 50 years old. With this qPCR assay it will be possible to trace the presence of G. semen in environments prior to the onset of algae-specific monitoring programs as well as for quantification in water column samples.

AimTo test if a phytoplankton bloom is panmictic, or whether geographical and environmental factors cause spatial and temporal genetic structure.LocationBaltic Sea.MethodDuring four cruises, we iso ...

6 pages, 2 figures, supporting information https://doi.org/10.1111/jpy.12748, 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, The work was supported through the Russian Foundation for Basic Research (project # 16-04-01704) to NA and a Swedish Research Council grant (2012-10-24) to KR as well as the marine microbial eukaryote transcriptome sequencing project (MMETSP) of the Gordon and Betty Moore Foundation

Special Issue: EMI is 20!.-- 10 pages, 3 figures, 1 table, supporting information https://doi.org/10.1111/1462-2920.14265, 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, This project was supported by the Swedish Research Council grants 349–2007-8690 (CAnMove) and 621–2012-3726 (to KR). RL was supported by a Ramón y Cajal fellowship (RYC-2013–12554, MINECO, Spain)

Colonization of new habitats through dispersal of phytoplankton cysts might be limited, if resident populations outcompete invaders during germination. We reciprocally transferred Gonyostomum semen (Raphidophyceae) cysts from three lakes into native and foreign waters originating from the respective habitats. Germination rate and germling growth were impacted by water origin, but there was no preference for native water. Gonyostomum semen's ability to germinate in different conditions might explain its expansion in northern Europe.

Many recent studies have found genetically differentiated populations in microorganisms despite potentially high dispersal. We designed a study to specifically examine the importance of physical dispersal barriers, i.e. geographic distance and lack of hydrological connectivity, in restricting gene flow and enhancing divergence in limnic microorganisms. We focused on the nuisance microalga Gonyostomum semen, which has recently expanded in northern Europe and differentiated into genetically distinct populations. Gonyostomum semen was sampled from six lakes distributed in two adjacent watersheds, which thereby comprised, both connected and non-connected lakes. The individual isolates were genotyped by Amplified Fragment Length Polymorphism. Several lake populations were differentiated from each other, but connectivity within watersheds could not explain the observed population genetic pattern. However, isolation by distance was moderate and might limit the gene flow among distant populations. In addition, we found low, but significant linkage disequilibrium, which indicates regular sexual recombination in this species, despite its high degree of asexual reproduction. Therefore, we conclude that the genetic properties of microalgae with occasional sexual reproduction essentially mirror regularly recombining species. Furthermore, the data indicated bottlenecks supporting the hypothesized recent range expansion of this species. (Less)

Processes of rapid radiation among unicellular eukaryotes are much less studied than among multicellular organisms. We have investigated a lineage of cold-water microeukaryotes (protists) that appear to have diverged recently. This lineage stands in stark contrast to known examples of phylogenetically closely related protists, in which genetic difference is typically larger than morphological differences. We found that the group not only consists of the marine-brackish dinoflagellate species Scrippsiella hangoei and the freshwater species Peridinium aciculiferum as discovered previously but also of a whole species flock. The additional species include Peridinium euryceps and Peridinium baicalense, which are restricted to a few lakes, in particular to the ancient Lake Baikal, Russia, and freshwater S. hangoei from Lake Baikal. These species are characterized by relatively large conspicuous morphological differences, which have given rise to the different species descriptions. However, our scanning electron microscopic studies indicate that they belong to a single genus according to traditional morphological characterization of dinoflagellates (thecal plate patterns). Moreover, we found that they have identical SSU (small subunit) rDNA fragments and distinct but very small differences in the DNA markers LSU (large subunit) rDNA, ITS2 (internal transcribed spacer 2) and COB (cytochrome b) gene, which are used to delineate dinoflagellates species. As some of the species co-occur, and all four have small but species–specific sequence differences, we suggest that these taxa are not a case of phenotypic plasticity but originated via recent adaptive radiation. We propose that this is the first clear example among free-living microeukaryotes of recent rapid diversification into several species followed by dispersion to environments with different ecological conditions.

A B S T R A C T Recent studies show that both marine and limnic microalgal species often consist of several genetically distinct populations. This is also valid for the nuisance freshwater algae Gonyostomum semen, which originates from acidic, brown water swamp lakes, but can nowadays also be found in clearer lakes with close to neutral pH. We hypothesized that the observed genetic differentiation among G. semen lake populations, reported in earlier studies, is connected to adaptation to local environmental conditions. In the present study we performed controlled laboratory experiments to test whether 12 strains originating from five lakes varied in their response to five to six different pHs, light intensities and DOC concentrations. Overall, growth (0.01–0.37 day � 1 ) was observed over a wide range of light intensities and pHs, demonstrating high potential for photoacclimation and extensive plasticity of individual strains. Moreover, we found similar growth rates and consistent growth optima for specific pHs by strains from the same lake, suggesting genetic differentiation of populations into distinct phenotypes. However, observed strain specific preferences did not always reflect environmental conditions in the lakes of origin and provided limited evidence for the hypothesized local adaptation. Instead, the observed phenotypic differentiation may indicate resilient effects of founder events. We suggest that the wide phenotypic plasticity in this species enables it to thrive in fluctuating and variable environments, and may play a role in its ability to colonize new habitats.

Whether or not communities of microbial eukaryotes are structured in the same way as prokaryotes is a basic 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 6,000 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 environmental selection driven by a salinity gradient in prokaryotes and ecological drift in microeukaryotes. Nevertheless, we detected also a number of microeukaryotes with specific responses to salinity, indicating that albeit minor, environmental selection has had a role in the assembly of their communities. Altogether, we conclude that habitat diversification can promote contrasting responses in planktonic microeukaryotes and prokaryotes belonging to the same communities.

Aquatic habitats are usually structured by light attenuation with depth resulting in different microalgal communities, each one adapted to a certain light regime by their specific pigment composition. Several taxa contain pigments restricted to one phylogenetic group, making them useful as marker pigments in phytoplankton community studies. The nuisance and invasive freshwater microalga Gonyostomum semen (Raphidophyceae) is mainly found in brown water lakes with sharp vertical gradients in light intensity and color. However, its pigment composition and potential photoadaptations have not been comprehensively studied. We analyzed the photopigment composition of 12 genetically different strains of G. semen by high performance liquid chromatography after acclimation to different light conditions. We confirmed the pigments chl a, chl c1c2, diadinoxanthin, trans-neoxanthin, cis-neoxanthin, α and β carotene, which have already been reported for G. semen. In addition, we identified, for the first time, the pigments violaxan-thin, zeaxanthin, and alloxanthin in this species. Alloxanthin has never been observed in raphidophytes before, suggesting differences in evolutionary plastid acquisition between freshwater lineages and the well-described marine species. The amount of total chl a per cell generally decreased with increasing light intensity. In contrast, the increasing ratios of the prominent pigments diadinoxanthin and alloxanthin per chl a with light intensity suggest photoprotective functions. In addition, we found significant variation in cell-specific pigment concentration among strains, grouped by lake of origin, which might correspond to genetic differences between strains and populations.

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.

The saline lakes of the Vestfold Hills in Antarctica offer a remarkable natural laboratory where the adaptation of planktonic protists to a range of evolving physiochemical conditions can be investigated. This study illustrates how an ancestral marine community has undergone radical simplification leaving a small number of well-adapted species. Our objective was to investigate the species composition and annual dynamics of dinoflagellate communities in three saline Antarctic lakes. We observed that dinoflagellates occur year-round despite extremely low PAR during the southern winter, which suggests significant mixotrophic or heterotrophic activity. Only a small number of dominant dinoflagellate species were found in each lake, in contrast to the species-rich Southern Ocean from which the lake communities are believed to be derived. We verified that the lake species were representatives of the marine polar dinoflagellate community, and not freshwater species. Polarella glacialis Montresor, Procaccini et Stoecker, a bipolar marine species, was for the first time described in a lake habitat and was an important phototrophic component in the higher salinity lakes. In the brackish lakes, we found a new sibling species to the brackish-water species Scrippsiella hangoei (J. Schiller) J. Larsen, previously observed only in the Baltic Sea.

Resting stages (e.g. cysts) play an important role in the life history and ecology of phytoplankton, e.g. the survival, reproduction, genetic recombination, and dispersal of many species. Marine dinoflagellates cysts have been intensively studied by both geologists and biologists, but freshwater cysts have received less attention. There are approximately 350 freshwater dinoflagellate species, and resting cysts have been described for 84 species. We evaluated the descriptions, and we reproduced images for each cyst type. The review highlighted the importance of cyst characters for taxonomy and phylogeny. We suggested that shape, wall ornamentation and possibly the archeopyle and color were important morphological characteristics at the generic level and above. The ecology of freshwater dinoflagellate cysts was reviewed, and the ecological role of cysts was discussed. The potential of freshwater cysts for Quaternary palaeoecological reconstructions was highlighted, revealing that these could serve as useful indicators of temperature, pH and productivity.

Gonyostomum semen (Ehrenb) Diesing is a bloom-forming and noxious phytoplankton species, that usually occurs in brown-water lakes and which is often referred to as an invasive species. The aim of our study was to analyze changes over time in the occurrence and distribution of blooms, and to find possible drivers of this change. We also performed spatial analyses to identify environmental factors coupled to Gonyostomum's distribution. The effect of temperature on key processes in the Gonyostomum life cycle was further investigated experimentally to determine potential mechanistic causes. Our results show that G. semen has expanded in Swedish lakes since 1988. At the turn of the Millennium it was present in more than a quarter of the lakes included in the Swedish national lake monitoring program. Gonyostomum-lakes have significantly higher DOC, higher nutrient levels, and lower pH than non-Gonyostomum lakes. Trend analyses show a significant increase in the number of lakes with Gonyostomum, as well as in biomass and occurrence in samples. One explanation is that we more often find water temperatures exceeding 6 degrees C, which is also the threshold for positive growth in our laboratory experiments. Moreover, according to our partial least square regression model (PLS) analysis in one lake, we find that the increase in biomass is a function of temperature in combination with other factors. Thus, we conclude that an increase in water temperature resulting in longer growth season may be a driver of the expansion of Gonyostomum. However, temperature alone cannot explain why the species has expanded to new lakes within the same climatic region. Possibly an interplay between DOC and temperature can explain the patterns observed. (C) 2012 Elsevier B.V. All rights reserved. (Less)

The nuisance alga Gonyostomum semen (Raphidophyceae) has expanded in the Nordic countries during the last decades and can dominate lake phytoplankton communities almost completely. A possible explanation to its dominance could be limited grazing by zooplankton. We investigated the potential grazing pressure on G. semen using an experimental approach supported by field data. We determined the grazing rate by cladocerans, calanoid copepods, and Chaoborus larvae to determine which were able to feed on G. semen. Only the large cladoceran Daphnia magna was able to feed successfully on G. semen. The large cell size of G. semen was likely a limiting factor for the filtering apparatus of smaller cladocerans. The copepod Eudiaptomus gracilis did not graze on G. semen, although the mechanism behind this selective feeding is still unknown. In addition to the experimental study, we quantified the zooplankton and phytoplankton communities in 40 lakes to determine the composition and abundance of the zooplankton communities co-occurring with G. semen, suggesting that large cladoceran species were not present in lakes where G. semen occurred. Hence, the growth of G. semen is not significantly controlled by grazing in natural systems, which likely facilitates bloom formation and invasion success of G. semen.

We tested the effect of the cyanobacterium Gloeotrichia echinulata on a diverse array of phytoplankton. We found that Gloeotrichia increased the growth rates of five of seven phytoplankton species up to 620% in comparison with a medium-only control after 96 h.

Until recently, the evolutionary relationships between marine and freshwater microbes were unclear, but the use of molecular phylogenies is beginning to shed light on this subject. An increasing amount of studies are showing that marine and freshwater microbes (including viruses) are usually not closely related, often grouping into distinct marine and freshwater phylogenetic clusters, similar to what has been reported before for macroorganisms. These studies indicate that marine-freshwater transitions have been infrequent events during the diversification of microbes and that most of these transitions occurred a long time ago in evolutionary terms. Here we discuss the significance of recent studies addressing this question and consider possible avenues for future research.

The relationships among cellular nutrient status, environmental conditions (temperature and nutrient availability), and cyst production were studied in batch cultures of three cold-water dinoflagellates (Scrippsiella hangoei, Gymnodinium corollarium, and Woloszynskia halophila) isolated from the Baltic Sea. We tested the effect of increasing temperature while providing nutrient-replete conditions as well as the effect of ambient nutrient (N, P) deficiency. The results revealed different encystment cues and patterns in the three species. While depletion of ambient nitrogen and subsequent internal N stress were the primary factors behind cyst production of G. corollarium, higher temperature led to substantial encystment of S. hangoei and W. halophila without a direct link to cellular nutrient physiology. In W. halophila, N limitation induced a transition of the population to small cells presumably representing gametes, but this process was not followed by cyst formation. Phosphorus stress was not directly linked to cyst formation in any of the species. Our data indicate that both reliable token cues (such as temperature) and ultimate causes (for example, nutrient depletion) for encystment are likely involved in the cystformation process. Such duality might provide an explanation for multiple triggers inducing encystment in laboratory settings and the lack of evidence for a direct relationship between nutrient depletion and cyst formation in the field.

The saline lakes of the Vestfold Hills in Antarctica offer a remarkable natural laboratory where the adaptation of planktonic protists to a range of evolving physiochemical conditions can be investigated. This study illustrates how an ancestral marine community has undergone radical simplification leaving a small number of well-adapted species. Our objective was to investigate the species composition and annual dynamics of dinoflagellate communities in three saline Antarctic lakes. We observed that dinoflagellates occur year-round despite extremely low PAR during the southern winter, which suggests significant mixotrophic or heterotrophic activity. Only a small number of dominant dinoflagellate species were found in each lake, in contrast to the species-rich Southern Ocean from which the lake communities are believed to be derived. We verified that the lake species were representatives of the marine polar dinoflagellate community, and not freshwater species. Polarella glacialis Montresor, Procaccini et Stoecker, a bipolar marine species, was for the first time described in a lake habitat and was an important phototrophic component in the higher salinity lakes. In the brackish lakes, we found a new sibling species to the brackish-water species Scrippsiella hangoei (J. Schiller) J. Larsen, previously observed only in the Baltic Sea.

We have constructed extensive 18S-28S rDNA dinoflagellate phylogenies (>200 sequences for each marker) using Maximum Likelihood and Bayesian Inference to study the evolutionary relationships among marine and freshwater species (43 new sequences). Our results indicated that (a) marine and freshwater species are usually not closely related, (b) several freshwater species cluster into monophyletic groups, (c) most marine-freshwater transitions do not seem to have occurred recently and, (d) only a small fraction of the marine lineages seem to have colonized fresh waters. Thus, it becomes apparent that the marine-freshwater boundary has acted as a barrier during the evolutionary diversification of dinoflagellates. Our results also shed light on the phylogenetic positions of several freshwater dinoflagellates which, to date, were uncertain.

The dinoflagellate Peridinium aciculiferum forms blooms underneath the ice in temperate lakes and has previously been shown to have an allelopathic effect on a natural competitor as well as being haemolytic. Hence, we investigated whether P. aciculiferum is allelopathic towards a wide range of different freshwater phytoplankton species. We also tested the hypothesis that nutrient (N and P) deficiency enhances its allelopathic effect. In addition, we explored how target cell density affected the extent of the allelopathic effect. Our findings showed that P. aciculiferum had a negative effect (mortality through lyses) on Synura petersenii (Chrysophyceae), Peridinium inconspicuum (Dinophyceae), Cyclotella sp. (Bacillariophyceae), Cryptomonas sp. and Rhodomonas lacustris (Cryptophyceae). Only Chlamydomonas reinhardtii (Chlorophyceae) cells were not negatively affected. In semi-continuously grown nutrient deficient cultures, we detected no allelopathic activity, in contrast to the findings for batch cultures. ...

Colonization of new habitats through dispersal of phytoplankton cysts might be limited, if resident populations outcompete invaders during germination. We reciprocally transferred Gonyostomum semen (Raphidophyceae) cysts from three lakes into native and foreign waters originating from the respective habitats. Germination rate and germling growth were impacted by water origin, but there was no preference for native water. Gonyostomum semen's ability to germinate in different conditions might explain its expansion in northern Europe.

Dinoflagellate successional strategies and community structure were investigated in Perch Pond, a temperate estuary on the North American east coast by field surveys as well as laboratory investigations on growth rates, cyst maturation period, and cyst germination temperature thresholds. The dominant species were those predicted by the Smayda and Reynolds Rules of Assembly life form model. Three successional strategies were characterized: (1) holoplanktonic, (2) meroplanktonic (i.e., germinated from cysts), and (3) introduced by advection. The seasonal succession of the meroplanktonic dinoflagellates that were studied reflects the differential lengths of their mandatory dormancy periods as well as differences in their temperature thresholds or "windows" for germination. The holoplanktonic species present at low densities year-round in Perch Pond had a wide temperature tolerance for growth and thus did not need a cyst stage to survive seasonal extremes. Another non-cyst-forming species relied solely on advection to inoculate the salt pond; thus, blooms in successive years would be expected to be more stochastic in nature than for the other two strategies. The timing of cyst formation and population decline for meroplanktonic species corresponded on several occasions to an increase in grazers, suggesting that grazing might have contributed to bloom decline from cyst formation. This timing also suggests the possibility of encystment as a predator avoidance strategy. We suggest that seasonal succession of cyst-forming dinoflagellates is not stochastic. Instead, the appearance of these species in the plankton is predictable on the basis of measurable physiological responses to both endogenous and exogenous factors that they experience during dormancy and quiescence.

We investigated whether phytoplankton communities in two lakes in SW Greenland were phosphorus or nitrogen limited. The study lakes have contrasting water chemistry (mean conductivities differ ten fold) and are located near Kangerlussuaq, SW Greenland (~67°N, 51°W). A microcosm nutrient enrichment experiment was performed in June 2003 to determine whether nitrate or phosphate addition stimulated phytoplankton growth. Samples were analysed for species composition, biomass, and alkaline phosphatase activity (APA). Initially, both lakes had extremely low total phosphorus but high total nitrogen concentrations and high APA, suggesting that the phytoplankton were phosphorus limited prior to the start of the experiment. The phytoplankton composition and biomass (mainly Ochromonas spp.) responded to phosphate but not to nitrate addition. In both lakes, chlorophyll a increased significantly when phosphate was added. Furthermore, APA was significantly lower in the two lakes when phosphate was added compared to the control and the nitrogen addition treatment. The dominance of mixotrophic phytoplankton and high DOC values suggest that these lakes may be regulated by microbial loop processes.

Adaptations and, counteradaptations are common in coevolving predatorprey systems, but little is known of the role of maternal transfer of adaptive traits in mediating species interactions. Here, we focused on tolerance against cyanobacterial toxins and asked whether this tolerance was an induced defense developed during Daphnia's lifetime, whether it was a trait that is constantly expressed, and whether such tolerance to the toxin can be transferred to the next generation through maternal effects. These questions were addressed by feeding a single clone of Daphnia magna a diet with and without algal toxin and recording changes in fitness (as intrinsic rate of population increase). Analysis of F1, F2, and F3 generations revealed that the increased tolerance to toxic Microcystis was an inducible defense developed during an individual's lifetime, and that this trait could be transferred from mother to offspring. This maternal effect was expressed in several fitness parameters, including shorter time to maturity and first reproduction, and higher numbers of offspring compared to inexperienced individuals. In some circumstances, such maternal effects may increase population production by up to 40% and may help to stabilize material and energy transfer to higher trophic levels.