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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.

DNA can be preserved in marine and freshwater sediments both in bulk sediment and in intact, viable resting stages. Here, we assess the potential for combined use of ancient, environmental, DNA and timeseries of resurrected long-term dormant organisms, to reconstruct trophic interactions and evolutionary adaptation to changing environments. These new methods, coupled with independent evidence of biotic and abiotic forcing factors, can provide a holistic view of past ecosystems beyond that offered by standard palaeoecology, help us assess implications of ecological and molecular change for contemporary ecosystem functioning and services, and improve our ability to predict adaptation to environmental stress., Ellegaard et al. discuss the potential for using ancient environmental DNA (eDNA), combined with resurrection ecology, to analyse trophic interactions and evolutionary adaptation to changing environments. Their Review suggests that these techniques will improve our ability to predict genetic and phenotypic adaptation to environmental stress.

The salinity gradient separating marine and freshwater environments represents a major ecological divide for microbiota, yet the mechanisms by which marine microbes have adapted to and ultimately diversified in freshwater environments are poorly understood. Here, we take advantage of a natural evolutionary experiment: the colonization of the brackish Baltic Sea by the ancestrally marine diatom Skeletonema marinoi. To understand how diatoms respond to low salinity, we characterized transcriptomic responses of S. marinoi grown in a common garden. Our experiment included eight genotypes from source populations spanning the Baltic Sea salinity cline. Changes in gene expression revealed a shared response to salinity across genotypes, where low salinities induced profound changes in cellular metabolism, including upregulation of carbon fixation and storage compound biosynthesis, and increased nutrient demand and oxidative stress. Nevertheless, the genotype effect overshadowed the salinity effect, as genotypes differed significantly in their response, both in the magnitude and direction of gene expression. Intraspecific differences included regulation of transcription and translation, nitrogen metabolism, cell signaling, and aerobic respiration. The high degree of intraspecific variation in gene expression observed here highlights an important but often overlooked source of biological variation associated with how diatoms respond and adapt to environmental change.

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.

Special issue Marine Benthic Harmful Algal Blooms.-- 11 pages, 5 figures, supplementary materials https://doi.org/10.1016/j.hal.2022.102352.-- Data availability: Data will be made available on request, A new chytrid genus and species was isolated and cultured from samples obtained in the Baltic Sea during a dinoflagellate bloom event. This species is characterized by having a spherical sporangium without papillae and zoospores of 2–3 µm in diameter that are released through 3 discharge pores. Molecular phylogeny based on ribosomal operon showed its sister position to the Dinomyces cluster in Rhizophydiales. Zoospores lack fenestrated cisternae but contain a paracrystalline inclusion, found in a Rhizophydiales representative for the first time. Additionally, the kinetid features are uncommon for Rhizophydiales and only observed in Dinomyces representatives so far. These morphological features and its phylogenetic relationships justify the description of the new genus and speciesParadinomyces triforaminorum gen. nov. sp. nov. belonging to the family Dinomycetaceae. The chytrid was detected during a high-biomass bloom of the dinoflagellate Kryptoperidinium foliaceum. Laboratory experiments suggest this species is highly specific and demonstrate the impact it can have on HAB development. The chytrid co-occurred with three other parasites belonging to Chytridiomycota (Fungi) and Perkinsea (Alveolata), highlighting that parasitic interactions are common during HABs in brackish and marine systems, and these multiple parasites compete for similar hosts, AR and EG were funded by the Spanish MICINN Project SMART PID2020-112978GB-I00. This work was partly supported by the Russian Science Foundation grant no. 21–74–20089 (writing, microscopy analysis), the infrastructure of SAK's laboratory was supported by ZIN RAS program 1021051402849–1, and the C7 strain was incorporated in CCPP ZIN RAS collection supported by a grant 075–15–2021–1069 of the Ministry of Science and Higher Education of the Russian Federation. AK was supported by the Academy of Finland, grant 310,449. MK was supported by JSPS KAKENHI 19H05667. EA was supported by The Royal Society (UK) under the Newton International Fellowship (grant number NF170346, call 2017), With the institutional support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S)

In seasonal environments, strong gradients of environmental parameters can shape life cycles of phytoplankton. Depending on the rate of environmental fluctuation, specialist or generalist strategies may be favored, potentially affecting life cycle transitions. The present study examined life cycle transitions of the toxin producing Baltic dinoflagellate Alexandrium ostenfeldii and their regulation by environmental factors (temperature and nutrients). This investigation aimed to determine whether genetic recombination of different strains is required for resting cyst formation and whether newly formed cysts are dormant. Field data (temperature and salinity) and sediment surface samples were collected from a site with recurrent blooms and germination and encystment experiments were conducted under controlled laboratory conditions. Results indicate a lack of seasonal germination pattern, set by an endogenous rhythm, as commonly found with other dinoflagellates from the Baltic Sea. Germination of quiescent cysts was triggered by temperatures exceeding 10°C and combined nutrient limitation of nitrogen and phosphorus or a drop in temperature from 16 to 10°C triggered encystment most efficiently. Genetic recombination was not mandatory for the formation of resting cysts, but supported higher numbers of resistant cysts and enhanced germination capacity after a resting period. Findings from this study confirm that A. ostenfeldii follows a generalist germination and cyst formation strategy, driven by strong seasonality, which may support its persistence and possibly expansion in marginal environments in the future, if higher temperatures facilitate a longer growth season.

Due to its crucial role in the ecosystem, phytoplankton is incorporated in marine ecosystem models. Most models however neglect the evolutionary potential of phytoplankton. Previous resurrection experiments with a spring bloom dinoflagellate suggest that the past century of global warming has caused an adaptive response in an important life cycle trait, the encystment rate. Here, based on this resurrection case study, we apply an advanced ecosystem model including selection and mutation, to test whether a temperature increase could induce a change in encystment. In line with the findings from resurrection experiments, our results show that in warmer waters strains with a lower encystment rate benefit over those with a higher encystment rate. The magnitude of change in encystment rate is however only reproduced, if additional factors, like eutrophication and a cyst mortality that increases with temperature, are considered. By using this ecosystem model including selection and mutation, we demonstrate that ecosystem modeling represents a powerful approach to investigate the adaptive potential of phytoplankton.

Environmental conditions regulate the germination of phytoplankton resting stages. While some factors lead to synchronous germination, others stimulate germination of only a small fraction of the resting stages. This suggests that habitat filters may act on the germination level and thus affect selection of blooming strains. Benthic ?seed banks? of the toxic dinoflagellate Alexandrium ostenfeldii from the Baltic Sea are genetically and phenotypically diverse, indicating a high potential for adaptation by selection on standing genetic variation. Here, we experimentally tested the role of climate-related salinity and temperature as selection filters during germination and subsequent establishment of A. ostenfeldii strains. A representative resting cyst population was isolated from sediment samples, and germination and reciprocal transplantation experiments were carried out, including four treatments: Average present day germination conditions and three potential future conditions: high temperature, low salinity, and high temperature in combination with low salinity. We found that the final germination success of A. ostenfeldii resting cysts was unaffected by temperature and salinity in the range tested. A high germination success of more than 80% in all treatments indicates that strains are not selected by temperature and salinity during germination, but selection becomes more important shortly after germination, in the vegetative stage of the life cycle. Moreover, strains were not adapted to germination conditions. Instead, highly plastic responses occurred after transplantation and significantly higher growth rates were observed at higher temperature. High variability of strain-specific responses has probably masked the overall effect of the treatments, highlighting the importance of testing the effect of environmental factors on many strains. It is likely that A. ostenfeldii populations can persist in the future, because suitable strains, which are able to germinate and grow well at potential future climate conditions, are part of the highly diverse cyst population. OPEN RESEARCH BADGES This article has been awarded Open Data badge. All materials and data are publicly accessible via the Open Science Framework at https://doi.org/10.5061/dryad.c8c83nr. Learn more about the Open Practices badges from the Center for Open Science: https://osf.io/tvyxz/wiki. peerReviewed

In recent years, blooms of toxic Alexandrium ostenfeldii strains have been reported from around the world. In 2013, the species formed a red tide in a shallow lagoon in western Japan, which was the first report of the species in the area. To investigate the genetic relatedness of Japanese A. ostenfeldii and global isolates, the full-length SSU, ITS and LSU sequences were determined, and phylogenetic analyses were conducted for isolates from western and northern Japan and from the Baltic Sea. Genotyping and microsatellite sequence comparison were performed to estimate the divergence and connectivity between the populations from western Japan and the Baltic Sea. In all phylogenetic analyses, the isolates from western Japan grouped together with global isolates from shallow and low saline areas, such as the Baltic Sea, estuaries on the east coast of U.S.A. and from the Bohai Sea, China. In contrast, the isolates from northern Japan formed a well-supported separate group in the ITS and LSU phylogenies, indicating differentiation between the Japanese populations. This was further supported by the notable differentiation between the sequences of western and northern Japanese isolates, whereas the lowest differentiation was found between the western Japanese and Chinese isolates. Microsatellite genotyping revealed low genetic diversity in the western Japanese population, possibly explained by a recent introduction to the lagoon from where it was detected. The red tide recorded in the shallow lagoon followed notable changes in the salinity of the waterbody and phytoplankton composition, potentially facilitating the bloom of A. ostenfeldii.

Cyanobacteria of the order Nostocales, including Baltic Sea bloom-forming taxa Nodularia spumigena, Aphanizomenon flosaquae, and Dolichospermum spp., produce resting stages, known as akinetes, under unfavorable conditions. These akinetes can persist in the sediment and germinate if favorable conditions return, simultaneously representing past blooms and possibly contributing to future bloom formation. The present study characterized cyanobacterial akinete survival, germination, and potential cyanotoxin production in brackish water sediment archives from coastal and open Gulf of Finland in order to understand recent bloom expansion, akinete persistence, and cyanobacteria life cycles in the northern Baltic Sea. Results showed that cyanobacterial akinetes can persist in and germinate from Northern Baltic Sea sediment up to >40 and >400 years old, at coastal and open-sea locations, respectively. Akinete abundance and viability decreased with age and depth of vertical sediment layers. The detection of potential microcystin and nodularin production from akinetes was minimal and restricted to the surface sediment layers. Phylogenetic analysis of culturable cyanobacteria from the coastal sediment core indicated that most strains likely belonged to the benthic genus Anabaena. Potentially planktonic species of Dolichospermum could only be revived from the near-surface layers of the sediment, corresponding to an estimated age of 1–3 years. Results of germination experiments supported the notion that akinetes do not play an equally significant role in the life cycles of all bloom-forming cyanobacteria in the Baltic Sea. Overall, there was minimal congruence between akinete abundance, cyanotoxin concentration, and the presence of cyanotoxin biosynthetic genes in either sediment core. Further research is recommended to accurately detect and quantify akinetes and cyanotoxin genes from brackish water sediment samples in order to further describe species-specific benthic archives of cyanobacteria.

15 pages, 8 figures, 1 table, supplementary information https://doi.org/10.1007/s11557-020-01652-x, This is a post-peer-review, pre-copyedit version of an article published in Karpov, S.A., Reñé, A., Vishnyakov, A.E. et al. Parasitoid chytridiomycete Ericiomyces syringoforeus gen. et sp. nov. has unique cellular structures to infect the host. Mycol Progress 20:, 95-109 (2021). The final authenticated version is available online at: http://dx.doi.org/10.1007/s11557-020-01652-x, Many fungi have been identified as pathogens of marine algae. Among them, Chytridiomycota have been revealed as relatively highly abundant, but much of the diversity known within these groups is almost entirely based on environmental sequencing data. Here, we present a novel chytridiomycete genus and species, characterized by light microscopical observations, ultrastructure, and molecular phylogenetic analysis of the parasitic chytrid of brackish-water dinoflagellate Kryptoperidinium foliaceum from the Baltic Sea. Phylogenetic analysis of rDNA sequences and the ultrastructure of the strain reveals that it represents a new family in the order Rhizophydiales. Ericiomyces syringoforeus gen. et sp. nov. is a parasitoid with a life cycle composed by zoospores, which attach to the host, encyst, and produce a rhizoidal system (haustorium). Unlike typical Rhizophydiales chytrids, sporangium develops as a lateral outgrowth of the encysted zoospore. The ultrastructural study revealed at least two unique traits: the syringe-like organelle in the cyst, which supposed to paralyze the host, and funnel-shaped structure anchoring sporangium in the host wall. Sporangium matures and produces new zoospores within 3 days. Multiple infections are common and then the life cycle is 1–2 days shorter compared to the duration when a single infection occurred. Cross-infection experiments showed that E. syringoforeus could only infect dinoflagellates, being K. foliaceum highly susceptible to infection by the chytrid parasitoid. The effects of some fungal epidemics on populations of Kryptoperidinium are discussed, Light and electron microscopic studies and manuscript writing were supported by Russian Scientific Foundation grant 16-14-10302. SK and AV thank the Research Resource Centre for Molecular and Cell Technologies (RRC MCT) at St. Petersburg State University (SPbSU) for access to the EM facilities. EG, EA, and AR were supported by MINECO COPAS “Understanding top-down control in coastal bloom-forming protists” (CTM2017-86121-R). MK and KS were supported by JSPS KAKENHI grants 15KK0026 & 16H02943. AK and AP were supported by grant 251564 from Academy of Finland, With the funding support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI)

15 pages, 9 figures, 2 tables, supplementary materials https://doi.org/10.1016/j.hal.2020.101944, Perkinsea are a group of intracellular protist parasites that inhabit all types of aquatic environments and cause significant population declines of a wide variety of hosts. However, the diversity of this lineage is mostly represented by environmental rDNA sequences. Complete descriptions of Perkinsea that infect marine dinoflagellates have increased in recent literature due to the identification, isolation and culturing of representatives during bloom events, contributing to expand the knowledge on the diversity and ecology of the group. Shallow coastal areas in the Baltic Sea suffer seasonal dinoflagellate blooms. In summer 2016, two parasitoids were isolated during a Kryptoperidinium foliaceum bloom in the Baltic Sea. Morphological features and sequences of the small and large subunit of the ribosomal DNA gene revealed these two parasitoids were new species that belong to the genus Parvilucifera. This is the first time that Parvilucifera infections are reported in the Inner Baltic Sea. The first species, Parvilucifera sp. has some morphological and phylogenetic features in common with P. sinerae and P. corolla, although its ultrastructure could not be studied and the formal description could not be done. The second new species, named Parvilucifera catillosa, has several distinct morphological features in its zoospores (e.g. the presence of a rostrum), and in the shape and size of the apertures in the sporangium stage, which are larger and more protuberant than in the other species of the genus. Infections observed in the field and cross-infection experiments determined that the host range of both Parvilucifera species was restricted to dinoflagellates, each one showing a different host preference. The coexistence in the same environment by the two closely related parasitoids with very similar life cycles suggests that their niche separation is the preferred host, This study was supported by the Academy of Finland, grant 251564 to A.K. E. G and A.R. were supported by MINECO COPAS “Understanding top-down control in coastal bloom-forming protists” (CTM2017-86121-R). Phytoplankton cultures were provided by the culture collection of the Finnish Environment Institute (SYKE), With the funding support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI)

8 pages, 2 tables, A recently published study analyzed the phylogenetic relationship between the genera Centrodinium and Alexandrium, confirming an earlier publication showing the genus Alexandrium as paraphyletic. This most recent manuscript retained the genus Alexandrium, introduced a new genus Episemicolon, resurrected two genera, Gessnerium and Protogonyaulax, and stated that: “The polyphyly [sic] of Alexandrium is solved with the split into four genera”. However, these reintroduced taxa were not based on monophyletic groups. Therefore this work, if accepted, would result in replacing a single paraphyletic taxon with several non-monophyletic ones. The morphological data presented for genus characterization also do not convincingly support taxa delimitations. The combination of weak molecular phylogenetics and the lack of diagnostic traits (i.e., autapomorphies) render the applicability of the concept of limited use. The proposal to split the genus Alexandrium on the basis of our current knowledge is rejected herein. The aim here is not to present an alternative analysis and revision, but to maintain Alexandrium. A better constructed and more phylogenetically accurate revision can and should wait until more complete evidence becomes available and there is a strong reason to revise the genus Alexandrium. The reasons are explained in detail by a review of the available molecular and morphological data for species of the genera Alexandrium and Centrodinium. In addition, cyst morphology and chemotaxonomy are discussed, and the need for integrative taxonomy is highlighted, Support to DMA from the NOAA ECOHAB program (Grant #NA15NOS4780181) is gratefully acknowledged. Support to EG, AR, NS from the COPAs project (CTM2017-86121-R) is acknowledged. IGL and CJBS are COFFA-IPN and EDI-IPN fellows, With the funding support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI)

The history of expansion of bloom-forming cold water dinoflagellates in the Northern Baltic Sea was studied using 100-year-old sediment archives of their resting cysts. Vertical cyst distributions ...

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.

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

Dinoflagellate cyst abundance and species composition were investigated before, during and after the spring bloom in the Gulf of Finland, north-eastern Baltic Proper and Gulf of Riga in order to detect spatial and temporal dynamics. Transport of newly formed cysts by currents was modelled to explore the possible distance travelled by cysts before sedimentation. The cyst community of the spring bloom dinoflagellates was dominated by the cysts of Biecheleria baltica in all basins, despite its marginal value in the planktonic spring bloom community in the Gulf of Riga. Dinoflagellate cyst abundance in the surface sediments displayed temporal dynamics in all basins, however, this appeared to be also influenced by physical processes. The model simulation showed that newly formed cysts are transported around 10–30 km from the point of origin before deposited. The latter suggests that transport of resting stages in the water column significantly affects spatial cyst distribution in the sediments and thus needs to be considered in the interpretation of temporal biological productivity patterns of a water body from cyst proxies.

Sexual reproduction plays a fundamental role in diatom life cycles. It contributes to increasing genetic diversity through meiotic recombination and also represents the phase where large-sized cells are produced to counteract the cell size reduction process that characterizes these microalgae. With the aim to identify genes linked to the sexual phase of the centric planktonic diatom Skeletonema marinoi, we carried out an RNA-seq experiment comparing the expression level of transcripts in sexualized cells with that of large cells not competent for sex. A set of genes involved in meiosis were found upregulated. Despite the fact that flagellate gametes were observed in the sample, we did not detect the expression of genes involved in the synthesis of flagella that were upregulated during sexual reproduction in another centric diatom. A comparison with the set of genes changing during the first phases of sexual reproduction of the pennate diatom Pseudo-nitzschia multistriata revealed the existence of commonalities, including the strong upregulation of genes with an unknown function that we named Sex Induced Genes (SIG). Our results further broadened the panel of genes that can be used as a marker for sexual reproduction of diatoms, crucial for the interpretation of metatranscriptomic datasets.

The toxic dinoflagellate Alexandrium ostenfeldii is the only bioluminescent bloom-forming phytoplankton in coastal waters of the Baltic Sea. We analysed partial luciferase gene (lcf) sequences and bioluminescence production in Baltic A. ostenfeldii bloom populations to assess the distribution and consistency of the trait in the Baltic Sea, and to evaluate applications for early detection of toxic blooms. Lcf was consistently present in 61 Baltic Sea A. ostenfeldii strains isolated from six separate bloom sites. All Baltic Sea strains except one produced bioluminescence. In contrast, the presence of lcf and the ability to produce bioluminescence did vary among strains from other parts of Europe. In phylogenetic analyses, lcf sequences of Baltic Sea strains clustered separately from North Sea strains, but variation between Baltic Sea strains was not sufficient to distinguish between bloom populations. Clustering of the lcf marker was similar to internal transcribed spacer (ITS) sequences with differences being minor and limited to the lowest hierarchical clusters, indicating a similar rate of evolution of the two genes. In relation to monitoring, the consistent presence of lcf and close coupling of lcf with bioluminescence suggests that bioluminescence can be used to reliably monitor toxic bloom-forming A. ostenfeldii in the Baltic Sea.

Genetic diversity is considered an important factor, stabilizing ecological functions when organisms are faced with changing environmental conditions. Although well known from terrestrial systems, documentations of this relationship from marine organisms, and particularly planktonic microorganisms, are still limited. Here we experimentally tested the effects of genotypic diversity on ecologically relevant cellular parameters (growth, primary production, particulate organic carbon, particulate organic nitrogen, particulate organic phosphorus and biogenic silica) at optimal and suboptimal salinity conditions in a marine phytoplankton species. Multiple clonal genotyped and phenotypically characterized isolates of the diatom Skeletonema marinoi from the Baltic Sea were grown in monocultures and mixes of 5 and 20 clones at native (5 psu) and reduced (3 psu) salinities and respective parameters were compared. Re-genotyping of 30 individuals from each population at five microsatellite loci at the end of the experiment confirmed maintenance of genotypic richness. Although a diversity effect on growth was not detected, primary production and particulate organic nutrients were positively affected by increased diversity independent of salinity condition. Under salinity stress, highest values of primary production and particulate organic nitrogen content were measured at the high diversity level. The observed diversity effects emphasize the importance of genetic diversity of phytoplankton populations for ecological functions.

Gymnodimines are lipophilic toxins produced by the marine dinoflagellates Karenia selliformis and Alexandrium ostenfeldii. Currently four gymnodimine analogues are known and characterized. Here we describe a novel gymnodimine and a range of gymnodimine related compounds found in an A. ostenfeldii isolate from the northern Baltic Sea. Gymnodimine D (1) was extracted and purified from clonal cultures, and characterized by liquid chromatography–tandem mass spectrometry (LC–MS/MS), nuclear magnetic resonance (NMR) spectroscopy, and liquid chromatography–high resolution mass spectrometry (LC–HRMS) experiments. The structure of 1 is related to known gymnodimines (2–5) with a six-membered cyclic imine ring and several other fragments typical of gymnodimines. However, the carbon chain in the gymnodimine macrocyclic ring differs from the known gymnodimines in having two tetrahydrofuran rings in the macrocyclic ring.

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 ...

The Baltic Sea is affected by a range of human induced environmental pressures such as eutrophication. Here we synthesize the ongoing shift from diatom dominance toward more dinoflagellates in parts of the Baltic Sea during the spring bloom and its potential effects on biogeochemical cycling of key elements (e.g., C, N, and P). The spring bloom is the period with the highest annual primary production and sinking of organic matter to the sediment. The fate of this organic matter is a key driver for material fluxes, affecting ecosystem functioning and eutrophication feedback loops. The dominant diatoms and dinoflagellates appear to be functionally surrogates as both groups are able to effectively exhaust the wintertime accumulation of inorganic nutrients and produce bloom level biomass that contribute to vertical export of organic matter. However, the groups have very different sedimentation patterns, and the seafloor has variable potential to mineralize the settled biomass in the different sub-basins. While diatoms sink quickly out of the euphotic zone, dinoflagellates sink as inert resting cysts, or lyse in the water column contributing to slowly settling phyto-detritus. The dominance by either phytoplankton group thus directly affects both the summertime nutrient pools of the water column and the input of organic matter to the sediment but to contrasting directions. The proliferation of dinoflagellates with high encystment efficiency could increase sediment retention and burial of organic matter, alleviating the eutrophication problem and improve the environmental status of the Baltic Sea. This study was supported by the Academy of Finland (decisions no 259164 and 263376) and Estonian Research Council (no 1575P, KO). Further funding came from Walter and Andrée de Nottbeck Foundation (KS and ToT) and the Swedish Cultural Heritage (ToT). LT acknowledges support also from COCOA— Nutrient cocktail in coastal zones of the Baltic Sea—EU BONUS project. This study was supported by the Academy of Finland (decisions no 259164 and 263376) and Estonian Research Council (no 1575P, KO). Further funding came from Walter and Andrée de Nottbeck Foundation (KS and ToT) and the Swedish Cultural Heritage (ToT). LT acknowledges support also from COCOA— Nutrient cocktail in coastal zones of the Baltic Sea—EU BONUS project.

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

Selection of suitable genotypes from diverse seed banks may help phytoplankton populations to cope with environmental changes. This study examines whether the high genotypic diversity found in the Baltic cyst pool of the toxic dinoflagellate Alexandrium ostenfeldii is coupled to phenotypic variability that could aid short-term adaptation. Growth rates, cellular toxicities and bioluminescence of 34 genetically different clones isolated from cyst beds of four Baltic bloom sites were determined in batch culture experiments along temperature and salinity gradients covering present and future conditions in the Baltic Sea. For all parameters a significant effect of genotype on the response to temperature and salinity changes was identified. General or site-specific effects of the two factors remained minor. Clones thriving at future conditions were different from the best performing at present conditions, suggesting that genotypic shifts may be expected in the future. Increased proportions of highly potent saxitoxin were observed as a plastic response to temperature increase, indicating a potential for higher toxicity of future blooms. The observed standing variation in Baltic seed banks of A. ostenfeldii suggests that the population is likely to persist under environmental change.

Here it is reported the first detection of DV-chl a together with the usual chl a in the marine dinoflagellate Alexandrium ostenfeldii from the Baltic Sea. Growth response and photosynthetic parameters were examined at two irradiances (80 and 240 μmol photons m(-2) s(-1)) and temperatures (15 °C and 19 °C) in a divinylic strain (AOTV-OS20) versus a monovinylic one (AOTV-OS16), using in vivo chl a fluorescence kinetics of PSII to characterize photosynthetic parameters by pulse amplitude modulated fluorescence, (14)C assimilation rates and toxin analyses. The divinylic isolate exhibited slower growth and stronger sensitivity to high irradiance than normal chl a strain. DV-chl a : chl a ratios decreased along time (from 11.3 to < 0.5 after 10 months) and to restore them sub-cloning and selection of strains with highest DV-chl a content was required. A mutation and/or epigenetic changes in the expression of divinyl reductase gene/s in A. ostenfeldii may explain this altered pigment composition. Despite quite severe limitations (reduced fitness and gradual loss of DV-chl a content), the DV-chl a-containing line in A. ostenfeldii could provide a model organism in photosynthetic studies related with chl biosynthesis and evolution.

A saxitoxin (STX) proficiency test (PT) was organized as part of the Establishment of Quality Assurance for the Detection of Biological Toxins of Potential Bioterrorism Risk (EQuATox) project. The aim of this PT was to provide an evaluation of existing methods and the European laboratories' capabilities for the analysis of STX and some of its analogues in real samples. Homogenized mussel material and algal cell materials containing paralytic shellfish poisoning (PSP) toxins were produced as reference sample matrices. The reference material was characterized using various analytical methods. Acidified algal extract samples at two concentration levels were prepared from a bulk culture of PSP toxins producing dinoflagellate Alexandrium ostenfeldii. The homogeneity and stability of the prepared PT samples were studied and found to be fit-for-purpose. Thereafter, eight STX PT samples were sent to ten participating laboratories from eight countries. The PT offered the participating laboratories the possibility to assess their performance regarding the qualitative and quantitative detection of PSP toxins. Various techniques such as official Association of Official Analytical Chemists (AOAC) methods, immunoassays, and liquid chromatography-mass spectrometry were used for sample analyses.

Kolkwitziella acuta is a cyst-forming dinoflagellate with a unique tabulation, occurring in freshwater to brackish environments of Eurasia and the USA. Based on the unique thecal plate arrangement, this species was previously interpreted as a missing link in the evolution between the genus Protoperidinium and the Diplopsalioideae. We isolated living K. acuta cysts from the south-western Caspian Sea and the central Baltic (Finland), and re-established the cyst-theca relationship. The cysts had a distinctive polar and dorsoventral compression and a characteristic wrinkly texture. The plate formula of the thecate stage was Po, X, 4′, 2a, 7′′, 3c+t, ?s, 5′′′, 1′′′′. Large subunit (LSU) and small subunit (SSU) ribosomal DNA sequences obtained through single-cell PCR of these cysts reveal that this species is close to Protoperidinum excentricum which belongs to the Excentrica section of the genus Protoperidinium. We suggest that it evolved from the genus Diplopsalis through a split of the apical and anterior in...

Phytoplankton is the basis for aquatic food webs and mirrors the water quality. Conventionally, phytoplankton analysis has been done using time consuming and partly subjective microscopic observations, but next generation sequencing (NGS) technologies provide promising potential for rapid automated examination of environmental samples. Because many phytoplankton species have tough cell walls, methods for cell lysis and DNA or RNA isolation need to be efficient to allow unbiased nucleic acid retrieval. Here, we analyzed how two phytoplankton preservation methods, three commercial DNA extraction kits and their improvements, three RNA extraction methods, and two data analysis procedures affected the results of the NGS analysis. A mock community was pooled from phytoplankton species with variation in nucleus size and cell wall hardness. Although the study showed potential for studying Lugol-preserved sample collections, it demonstrated critical challenges in the DNA-based phytoplankton analysis in overall. The 18S rRNA gene sequencing output was highly affected by the variation in the rRNA gene copy numbers per cell, while sample preservation and nucleic acid extraction methods formed another source of variation. At the top, sequence-specific variation in the data quality introduced unexpected bioinformatics bias when the sliding-window method was used for the quality trimming of the Ion Torrent data. While DNA-based analyses did not correlate with biomasses or cell numbers of the mock community, rRNA-based analyses were less affected by different RNA extraction procedures and had better match with the biomasses, dry weight and carbon contents, and are therefore recommended for quantitative phytoplankton analyses. peerReviewed

Diatoms and dinoflagellates are major bloom-forming phytoplankton groups competing for resources in the oceans and coastal seas. Recent evidence suggests that their competition is significantly affected by climatic factors under ongoing change, modifying especially the conditions for cold-water, spring bloom communities in temperate and Arctic regions. We investigated the effects of phytoplankton community composition on spring bloom carbon flows and nutrient stoichiometry in multiyear mesocosm experiments. Comparison of differing communities showed that community structure significantly affected C accumulation parameters, with highest particulate organic carbon (POC) buildup and dissolved organic carbon (DOC) release in diatom-dominated communities. In terms of inorganic nutrient drawdown and bloom accumulation phase, the dominating groups behaved as functional surrogates. Dominance patterns, however, significantly affected C : N : P : Chl a ratios over the whole bloom event: when diatoms were dominant, these ratios increased compared to dinoflagellate dominance or mixed communities. Diatom-dominated communities sequestered carbon up to 3.6-fold higher than the expectation based on the Redfield ratio, and 2-fold higher compared to dinoflagellate dominance. To our knowledge, this is the first experimental report of consequences of climatically driven shifts in phytoplankton dominance patterns for carbon sequestration and related biogeochemical cycles in coastal seas. Our results also highlight the need for remote sensing technologies with taxonomical resolution, as the C : Chl a ratio was strongly dependent on community composition and bloom stage. Climate-driven changes in phytoplankton dominance patterns will have far-reaching consequences for major biogeochemical cycles and need to be considered in climate change scenarios for marine systems.

Alexandrium ostenfeldii is a globally distributed dinoflagellate species capable of producing several potent toxins. Blooms of this species are presently expanding, possibly as a result of changing environmental conditions or recent spreading of geographically distant populations. Population genetic analyses are a useful tool to investigate invasion and selection as potential mechanisms behind the global increase in toxic algal bloom events. In this study, we isolated ten polymorphic microsatellite markers from A. ostenfeldii by three techniques, two PCR-based techniques, and a next generation sequencing technique. To characterize the newly developed microsatellite loci, DNAs extracted from 40 clonal isolates of Baltic A. ostenfeldii were genotyped. The isolated microsatellite loci were shown to be sufficiently polymorphic; allele numbers ranged from 2 to 12 among the studied strains, and estimates of gene diversity varied between 0.10 and 0.92. The ten polymorphic genetic markers developed in this study will facilitate investigations on genetic structure and gene flow within and among A. ostenfeldii populations, as well as studies on the role of selection by changing environmental conditions in bloom formation.

The cryptomonad Rhinomonas nottbecki n. sp., isolated from the Baltic Sea, is described from live and fixed cells studied by light, scanning, and transmission electron microscopy together with sequences of the partial nucleus- and nucleomorph-encoded 18S rRNA genes as well as the nucleus-encoded ITS1, 5.8S, ITS2, and the 5′-end of the 28S rRNA gene regions. The sequence analyses include comparison with 43 strains from the family Pyrenomonadaceae. Rhinomonas nottbecki cells are dorsoventrally flattened, obloid in shape; 10.0–17.2 μm long, 5.5–8.1 μm thick, and 4.4–8.8 μm wide. The inner periplast has roughly hexagonal plates. Rhinomonas nottbecki cells resemble those of Rhinomonas reticulata, but the nucleomorph 18S rRNA gene of R. nottbecki differs by 2% from that of R. reticulata, while the ITS region by 11%. The intraspecific variability in the ITS region of R. nottbecki is 5%. In addition, the predicted ITS2 secondary structures are different in R. nottbecki and R. reticulata. The family Pyrenomonadaceae includes three clades: Clade A, Clade B, and Clade C. All Rhinomonas sequences branched within the Clade C, while the genus Rhodomonas is paraphyletic. The analyses suggest that the genus Storeatula is an alternating morphotype of the genera Rhinomonas and Rhodomonas and that the family Pyrenomonadaceae includes some species that were described multiple times, as well as novel species.

This study provides microscopic and molecular evidence for sexual reproduction in the homothallic centric diatom Skeletonema marinoi isolated from the Baltic Sea. The species is capable of restoring cell size asexually through an auxospore-like stage. However, cells were sexualized after shifting strains from low (6 PSU) to high (16 PSU) salinity. We observed flagellate male gametes and oogonia, with diameters of 3-4 and 3.2-6.3μm, respectively. Fertilization took place followed by the formation of round auxospores surrounded by thin siliceous incunabular scales. Auxosporulation was synchronized, and a maximum of auxospores was detected on day three following the salinity shift. The proportion of auxospores to vegetative cells ranged from 0.02 to 0.18. There was a significant correlation between auxosporulation success and inoculum cell density. At lower cell concentration (5,000 cells ml(-1)), proportionally fewer auxospores were formed. Auxospores were formed in single strains and in crosses of strains. The proportion of auxospores differed significantly among strains and crosses of strains. Additionally, we isolated single auxospores, obtained F1 strains and performed microsatellite based pedigree analysis of parental generations and their offspring. We proved that the auxospores were formed sexually, either by inter- or by intra-strain fertilization.

The paralytic shellfish toxin (PST) producing dinoflagellate Alexandrium ostenfeldii forms dense, recurrent blooms during summer in shallow coastal areas of the Baltic Sea. We studied the intra-population variability of its allelochemical potency and the responses of co-occurring and potentially competing dinoflagellates to the allelochemicals. The lytic activity of 10 northern Baltic A. ostenfeldii strains was evaluated by their EC 50 values (i.e. the cell concentration yielding a 50% decline in cryptophyte density), which were found to vary between 236 and 1726 cells ml −1 . When co-occurring dinoflagellates ( Kryptoperidinium foliaceum , Levanderina fissa and Heterocapsa triquetra ) were exposed to filtrate of A. ostenfeldii , short-term ( K. foliaceum cells formed cysts, L. fissa cells lost their cell shape and lysed, whereas H. triquetra cells shed their thecae. After 24 h, K. foliaceum had returned into vegetative cells and the number of immotile L. fissa and H. triquetra cells had significantly decreased. The results indicate that A. ostenfeldii can disturb the growth of competing dinoflagellates by excreting allelochemicals at bloom concentrations and that co-occurring species may develop efficient means to escape and recover from the allelochemicals, allowing them to coexist with A. ostenfeldii .