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

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

Decomposing multiple dimensions of stability in global change experiments.

Ecological stability is the central framework to understand an ecosystem's ability to absorb or recover from environmental change. Recent modelling and conceptual work suggests that stability is a multidimensional construct comprising different response aspects. Using two freshwater mesocosm experiments as case studies, we show how the response to single perturbations can be decomposed in different stability aspects (resistance, resilience, recovery, temporal stability) for both ecosystem functions and community composition. We find that extended community recovery is tightly connected to a nearly complete recovery of the function (biomass production), whereas systems with incomplete recovery of the species composition ranged widely in their biomass compared to controls. Moreover, recovery was most complete when either resistance or resilience was high, the latter associated with low temporal stability around the recovery trend. In summary, no single aspect of stability was sufficient to reflect the overall stability of the system.

Phylogeography of the freshwater raphidophyte Gonyostomum semen confirms a recent expansion in northern Europe by a single haplotype.

Gonyostmum semen is a freshwater raphidophyte that has increased in occurrence and abundance in several countries in northern Europe since the 1980s. More recently, the species has expanded rapidly also in north-eastern Europe, and it is frequently referred to as invasive. To better understand the species history, we have explored the phylogeography of G. semen using strains from northern Europe, United States, and Japan. Three regions of the ribosomal RNA gene (small subunit [SSU], internal transcribed spacer [ITS] and large subunit [LSU]) and one mitochondrial DNA marker (cox1) were analyzed. The SSU and partial LSU sequences were identical in all strains, confirming that they belong to the same species. The ITS region differentiated the American from the other strains, but showed high intra-strain variability. In contrast, the mitochondrial marker cox1 showed distinct differences between the European, American, and Japanese strains. Interestingly, only one cox1 haplotype was detected in European strains. The overall low diversity and weak geographic structure within northern European strains supported the hypothesis of a recent invasion of new lakes by G. semen. Our data also show that the invasive northern European lineage is genetically distinct from the lineages from the other continents. Finally, we concluded that the mitochondrial cox1 was the most useful marker in determining large-scale biogeographic patterns in this species.

Sterols of the green-pigmented, freshwater raphidophyte, Gonyostomum semen, from Scandinavian lakes.

Sterols are a class of membrane-reinforcing, ringed lipids which have a long history of examination in algae as a means of deriving chemotaxonomic relationships and as potential lipidic biomarkers. The Raphidophyceae represent a class of harmful, bloom-forming, marine and freshwater algae. To date, there have been four published examinations of their sterol composition, focusing primarily on brown-pigmented, marine species within the genera, Chattonella, Fibrocapsa, and Heterosigma. Lacking in these examinations has been the species Gonyostomum semen Ehrenb., which is a green-pigmented, freshwater raphidophyte with a worldwide distribution. The goal of this study was to examine the sterol composition of this nuisance alga, determine the potential of using its sterol profile as a biomarker, and finally to determine if there is any intraspecific variability between isolates. We have examined 21 isolates of G. semen from a number of Scandinavian lakes, and all were found to produce two major sterols, 24-ethylcholesta-5,22E-dien-3ß-ol and 24-ethylcholest-5-en-3ß-ol, and 24-methylcholest-5-en-3ß-ol as a minor sterol; the presence of 24-ethylcholesta-5,22E-dien-3ß-ol differentiates G. semen from brown-pigmented, marine raphidophytes which generally lack it. The results of this study indicate that isolates of G. semen from geographically separate lakes across Finland and Scandinavia have the same sterol biosynthetic pathway, and that there is no evolutionary divergence between the isolates with regard to sterol composition. The sterols of G. semen are not considered to be useful biomarkers for this particular organism because they are commonly found in other algae and plants.

Removal efficiency of a constructed wetland combined with ultrasound and UV devices for wastewater reuse in agriculture.

This study evaluates the treatment efficiency of a chemical-free water treatment for treating the secondary effluent of a municipal wastewater treatment plant with the aim of reusing the water for agriculture. Urban wastewater was treated by three units run in series: a full-scale horizontal sub-surface flow constructed wetland, a small pond with an ultrasound (US) system and a UV device. The treatment efficiency was evaluated in terms of the Italian wastewater limits for irrigation reuse, water quality improvement (removal percentage) and algae bloom control. The tolerable infection risk, associated with the use of wastewaters for irrigating crops, was also assessed by applying the microbial risk analyses proposed in the WHO guidelines for wastewater reuse. The constructed wetland was efficient in reducing physical-chemical and microbiological concentrations, and its efficiency was very steady over the investigation period. The UV system significantly improved water quality (p<0.05) in terms of pathogen concentration with a further average decrease from 0.35 to 1.23 log units, depending on the microbiological parameter. The US device was able to prevent algae bloom on a free water surface and maintain Chlorophyll-a concentration stable and low 2 months after activation.

Genetic diversity within and genetic differentiation between blooms of a microalgal species.

The field of genetic diversity in protists, particularly phytoplankton, is under expansion. However, little is known regarding variation in genetic diversity within populations over time. The aim of our study was to investigate intrapopulation genetic diversity and genetic differentiation in the freshwater bloom-forming microalga Gonyostomum semen (Raphidophyceae). The study covered a 2-year period including all phases of the bloom. Amplified fragment length polymorphism (AFLP) was used to determine the genetic structure and diversity of the population. Our results showed a significant differentiation between samples collected during the two blooms from consecutive years. Also, an increase of gene diversity and a loss of differentiation among sampling dates were observed over time within a single bloom. The latter observations may reflect the continuous germination of cysts from the sediment. The life cycle characteristics of G. semen, particularly reproduction and recruitment, most likely explain a high proportion of the observed variation. This study highlights the importance of the life cycle for the intraspecific genetic diversity of microbial species, which alternates between sexual and asexual reproduction.

High abundances of the nuisance raphidophyte Gonyostomum semen in brown water lakes are associated with high concentrations of iron.

Algal blooms occur frequently in lakes and oceans and the causes and consequences of those are often studied. In this study, we focus on a less well known type of algal bloom by the freshwater raphidophyte Gonyostomum semen. This species' abundance and occurrence is increasing, especially in brown water lakes, the most abundant lake type in the boreal zone. The aim of the study was to investigate which environmental factors are associated with G. semen by statistical evaluation of field data of 95 Swedish lakes over five years. Although we found G. semen to be associated with dark waters it was, contrary to our expectations, mainly high concentrations of iron, and only to a lesser extent high TOC (total organic carbon) concentrations, that were associated with blooms of G. semen. In addition, high phosphorus concentrations and low pH also appear to facilitate G. semen blooms. We suggest that browning of lakes caused by increased iron concentrations may decrease net heterotrophy by fostering heavy algal blooms, i.e. the opposite to commonly assumed effects of increased DOM (dissolved organic matter).

Distinct Oceanic Microbiomes From Viruses to Protists Located Near the Antarctic Circumpolar Current.

Microbes occupy diverse ecological niches and only through recent advances in next generation sequencing technologies have the true microbial diversity been revealed. Furthermore, lack of perceivable marine barriers to genetic dispersal (i.e., mountains or islands) has allowed the speculation that organisms that can be easily transported by currents and therefore proliferate everywhere. That said, ocean currents are now commonly being recognized as barriers for microbial dispersal. Here we analyzed samples collected from a total of six stations, four located in the Indian Ocean, and two in the Southern Ocean. Amplicon sequencing was used to characterize both prokaryotic and eukaryotic plankton communities, while shotgun sequencing was used for the combined environmental DNA (eDNA), microbial eDNA (meDNA), and viral fractions. We found that Cyanobacteria dominated the prokaryotic component in the South-West Indian Ocean, while γ-Proteobacteria dominated the South-East Indian Ocean. A combination of γ- and α-Proteobacteria dominated the Southern Ocean. Alveolates dominated almost exclusively the eukaryotic component, with variation in the ratio of Protoalveolata and Dinoflagellata depending on station. However, an increase in haptophyte relative abundance was observed in the Southern Ocean. Similarly, the viral fraction was dominated by members of the order Caudovirales across all stations; however, a higher presence of nucleocytoplasmic large DNA viruses (mainly chloroviruses and mimiviruses) was observed in the Southern Ocean. To our knowledge, this is the first that a statistical difference in the microbiome (from viruses to protists) between the subtropical Indian and Southern Oceans. We also show that not all phylotypes can be found everywhere, and that meDNA is not a suitable resource for monitoring aquatic microbial diversity.

Choice of molecular barcode will affect species prevalence but not bacterial community composition.

The rapid advancement of next generation sequencing protocols in recent years has led to the diversification in the methods used to study microbial communities; however, how comparable the data generated from these different methods are, remains unclear. In this study we compared the taxonomic composition and seasonal dynamics of the bacterial community determined by two distinct 16s amplicon sequencing protocols: sequencing of the V6 region of the 16s rRNA gene using 454 pyrosequencing vs the V4 region of the 16s rRNA gene using the Illumina Hiseq 2500 platform. Significant differences between relative abundances at all taxonomic levels were observed; however, their seasonal dynamics between phyla were largely consistent between methods. This study highlights that care must be taken when comparing datasets generated from different methods.

Population genetic structure of a microalgal species under expansion.

Biological invasions often cause major perturbations in the environment and are well studied among macroorganisms. Less is known about invasion by free-living microbes. Gonyostomum semen (Raphidophyceae) is a freshwater phytoplankton species that has increased in abundance in Northern Europe since the 1980's and has expanded its habitat range. In this study, we aimed to determine the genetic population structure of G. semen in Northern Europe and to what extent it reflects the species' recent expansion. We sampled lakes from 12 locations (11 lakes) in Norway, Sweden and Finland. Multiple strains from each location were genotyped using Amplified Fragment Length Polymorphism (AFLP). We found low differentiation between locations, and low gene diversity within each location. Moreover, there was an absence of genetic isolation with distance (Mantel test, p = 0.50). According to a Bayesian clustering method all the isolates belonged to the same genetic population. Together our data suggest the presence of one metapopulation and an overall low diversity, which is coherent with a recent expansion of G. semen.

Zooplankton feeding on the nuisance flagellate Gonyostomum semen.

The large bloom-forming flagellate Gonyostomum semen has been hypothesized to be inedible to naturally occurring zooplankton due to its large cell size and ejection of long slimy threads (trichocysts) induced by physical stimulation. In a grazing experiment using radiolabelled algae and zooplankton collected from lakes with recurring blooms of G. semen and lakes that rarely experience blooms, we found that Eudiaptomus gracilis and Holopedium gibberum fed on G. semen at high rates, whereas Daphnia cristata and Ceriodaphnia spp. did not. Grazing rates of E. gracilis were similar between bloom-lakes and lakes with low biomass of G. semen, indicating that the ability to feed on G. semen was not a result of local adaptation. The high grazing rates of two of the taxa in our experiment imply that some of the nutrients and energy taken up by G. semen can be transferred directly to higher trophic levels, although the predominance of small cladocerans during blooms may limit the importance of G. semen as a food resource. Based on grazing rates and previous observations on abundances of E. gracilis and H. gibberum, we conclude that there is a potential for grazer control of G. semen and discuss why blooms of G. semen still occur.